Caulobacter crescentus cells that display and secrete a self-interacting protein. This protein formed a de novo matrix and assembled cells into centimeter-scale ELMs. Discovery of design and assembly principles allowed us to tune the composition, mechanical properties, and catalytic function of these ELMs. This work provides genetic tools, design and assembly rules, and a platform for growing ELMs with control over both matrix and cellular structure and function.Engineered living materials (ELMs) embed living cells in a biopolymer matrix to create materials with tailored functions. While bottom-up assembly of macroscopic ELMs with a de novo matrix would offer the greatest control over material properties, we lack the ability to genetically encode a protein matrix that leads to collective self-organization. Here we report growth of ELMs from  Engineered living materials (ELMs) embed living cells in a biopolymer matrix to create novel materials with tailored functions. In this work, the authors engineered bacteria to grow novel macroscopic materials that can be reshaped, functionalized, and used to filter contaminated water while also showing that the stiffness of these materials can be tuned through genetic changes. Engineered living materials (ELMs)4 are inspired by naturally occurring living materials, but use synthetic biology to introduce tailored, non-natural functions. By incorporating engineered cells into a biopolymer matrix, these materials can function as living sensors5, therapeutics6, biomanufacturing platforms7, electronics8, energy converters9, and structural materials10. While cells confer functionality to ELMs, the matrix assembles the material and controls the bulk material composition, structure, and function11.Naturally occurring living biomaterials, such as bones or wood, grow bottom-up from a small number of progenitor cells into macroscale structures11 because secreting recombinant biopolymers at concentrations that gelate is challenging12 and because the assembly of micrometer-sized cells into centimeter-scale materials requires self-organization across length scales spanning four orders of magnitude. Engineering principles to achieve this assembly are unknown12. Therefore, most macroscopic ELMs have been produced by adopting a top-down approach (such as 3D printing) to incorporate living cells into an exogenous matrix14 or by processing microscopic ELMs that grow a synthetic biomolecular matrix into macroscopic materials19. The few autonomously produced, macroscopic ELMs have been created by genetically modifying existing nanocellulose matrices20 or genetically manipulating the mineralization of silica matrices. However, these two approaches to autonomously produced, macroscopic ELMs have afforded little genetic control over the mechanical properties, e.g., ~1.2–1.4-fold change in the storage modulus21. This tunability is much more limited than the tunability of naturally occurring materials, chemically synthesized materials, or macroscopic ELMs produced by processing23.Since the matrix plays such a key role in generating material properties, one primary goal of the field is to create ELMs that both have a synthetic biomolecular matrix—that can control these properties—and grow autonomously into macroscopic structures. However, such bottom-up, de novo ELMs are considered well beyond the current state-of- artEscherichia coli has been engineered to display interacting proteins, such as leucine zippers24 or antigen-nanobody pairs25, via outer membrane proteins. Engineered strains that display interacting pairs will self-assemble into cell–cell aggregates that flocculate25; however, these aggregates are microscopic and must be processed to form larger materials18. In contrast, micron-sized colloidal particles  that display DNA have been programmed to self-assemble into both microscopic21 and macroscopic crystalline solids26. Over two decades of work on these systems has established central principles that underlie their self-assembly27. One of these central principles is that the interactions between particles must be mediated by high-density surface modifications, e.g., 1 DNA molecule per 27 nm26. Since the outer membrane proteins used for bacterial adhesins are displayed at ~5% of this density, i.e., 1 nanobody per 640 nm28, we hypothesized that a matrix composed of self-interacting proteins displayed on bacteria at high density could lead to the formation of macroscopic solid materials.We posit that new strategies for developing synthetic biomolecular matrices to self-assemble bacteria into macroscopic ELMs can be informed by prior work on surface-engineered bacteria and surface-modified colloidal particles. The surface of Caulobacter crescentus for high-density peptide display29 and biopolymer secretion23. The S-layer forms a 2D crystalline layer on the extracellular surface of C. crescentus, opening the possibility of displaying proteins at a density of up to 1 protein per 70 nm30. Leveraging this prior work, here we describe the autonomous formation of macroscopic living material from C. crescentus engineered to display a synthetic, self-interacting, protein matrix based on the S-layer scaffold. We demonstrate that the mechanical properties of this material can be genetically controlled over a factor of ~25x. We also describe unexpected findings indicating that the protein matrix plays a multifaceted role in the material formation and that material assembly occurs through a multi-step process mediated by the air–water interface.We have previously engineered the surface layer (S-layer) of the oligotrophic bacterium C. crescentus31, we sought to create bottom-up ELMs composed of cells that interact at high density through a surface-bound, de novo matrix. To minimize native cell–cell interactions, we started with a C. crescentus background that lacks the adhesive holdfast and therefore cannot form a biofilm33; we refer to this as the wild-type strain. Next, we designed a displayed bottom-up de novo (BUD) protein by replacing the native copy of the surface layer (S-layer) RsaA30  based on human tropoelastin that contains 60 repeats of the Val-Pro-Gly-X-Gly motif 37 (ELP60). ELPs form elastic materials, are flexible, and self-associate in a concentration-dependent fashion. Thus, this region is designed to influence material properties, promote solution accessibility, and add non-covalent self-interactions. SpyTag38 was used as a functionalization tag, as it covalently binds to fusion proteins containing SpyCatcher. We also introduced a FLAG tag as an epitope marker. Lastly, the C-terminal domain of the BUD protein, consisting of the last 336 residues of RsaA, was chosen to mediate protein secretion29 and to self-associate39. We refer to this BUD protein-expressing strain of C. crescentus as the BUD-ELM strain. In this way, we designed a C. crescentus strain to display a high-density, surface-bound, elastin-based matrix across its entire surface  on all sides , and the BUD protein shows this same apparent difference in molecular weight (observed 102 vs. expected 86 kDa). Interestingly, we found secreted BUD protein in the medium under both static and shaking conditions  limit–90 ± 5% of cadmium was removed  to obtain the holo forms of the enzyme. After confirming the activity of holo GDH in both cases , which couples oxidation of glucose to the reduction of a soluble electron carrierier Fig. . This deIn summary, we developed macroscopic living materials that autonomously grow from engineered bacteria and that can be genetically encoded to have a wide range of mechanical properties. Specifically, we show that the expression of a self-interacting protein—the BUD protein—enables macroscopic material formation Fig. . When diE. coli with self-interacting proteins displayed at ~10% the density of our engineered C. crescentus strains lead to small cell–cell aggregates25. However, additional studies that systematically vary the surface density are needed to test this hypothesis. Another design rule that will be critical to understand and explore in future work is the nature and strength of the self-interactions in the BUD protein. We selected the RsaA690-1026 and ELP60 domains because prior reports demonstrate they can self-aggregate44. However, additional studies are needed to identify the nature of self-interactions and their strengths in the existing BUD protein and the range of self-interactions that permit the assembly of macroscopic materials.Our work identifies design rules that lead to the autonomous formation of BUD-ELMs and suggests other design rules to be tested. We identify a secreted matrix as a design constraint for this class of centimeter-scale, autonomously forming BUD-ELMs. Our work also indicates that a surface-anchored protein matrix is necessary for these materials to be cell-rich. Our data also suggests that this surface-anchored protein matrix may need to be present at high-density for cell-rich materials. This suggestion is supported by previous literature that shows that 18, we suggest that the use of the air–water interface to locally concentrate and order hydrophobic biomolecules into a matrix may represent a general assembly principle for macroscopic ELMs. The genetic tools and C. crescentus platform developed here will permit systematic exploration of design and assembly rules for programming the growth of centimeter-scale structures using living cells as building blocks.This work also identified assembly principles for the autonomous formation of macroscopic materials. We have demonstrated that nucleation of a pellicle at the liquid-air interface and hydrodynamically-driven coalescence and collapse of the pellicle are required to form macroscopic ELMs. Since pellicle formation is also a key step in nanocellulose-based living materialsC. crescentus BUD-ELM platform developed herein is the highly reproducible, autonomous formation of engineered living materials. Growing BUD-ELMs from an engineered strain of C. crescentus requires only control of the temperature, media composition, flask and culture volume, shaking speed, and shaking orbit. We envision this simplicity will enable the ready adoption of this platform by other researchers. The second advantage of this platform is that the modularity of the BUD protein and the ease of engineering protein biopolymers offer much greater opportunities for introducing desirable properties into the matrix11. The BUD-ELM variants described herein have storage moduli that ranges between 13 kPa, comparable to nanocellulose-based materials, and 0.5 kPa, comparable to printed curli fiber-based materials. Introducing sites for chemical crosslinking into the ELP domain could allow the BUD-ELMs to be developed into elastomers45. More broadly, this work enables leveraging known polypeptides and proteins that exhibit desirable optical, electrical, mechanical, thermal, transport, and catalytic properties46. We envision specific matrix properties that can be combined synergistically with existing cellular functions such as sensing, biomolecule production, and information processing. Thus, this work multiplies the opportunities to program ELMs tailored for applications in human health, energy, and the environment.By creating BUD-ELMs with a de novo, modular protein matrix, this work greatly expands the ability to tailor macroscopic ELMs for specific applications. One of the key advantages of the ∆ELP60—RCC004, and ∆SpyTag—RCC005) from the wild-type47 (MFm126), we cloned integration plasmids designed to facilitate the incorporation of synthetic DNA sequences into the rsaA locus using homologous recombination. The integration plasmid used to generate the original BUD-ELM strain (pSMCAF008) was cloned by inserting a target sequence into the multicloning site of the backbone plasmid pNPTS138 (GenBank: MK533795.1) using restriction enzymes (ApaI upstream and NheI downstream). The target sequence  encoded the ELP60-SpyTag flanked by 800 bp of homology regions up- and downstream of the native rsaA central domain (rsaA750–2073). The integration plasmids used to generate the ∆ELP60 and ∆SpyTag BUD-ELM strains  were cloned from the plasmid pSMCAF008 using Golden Gate assembly (PCR primers listed below).All strains and plasmids used in this work are listed in Supplementary Table 29: plasmid pSMCAF008, pSMCAF017 or pSMCAF018 was electroporated into E. coli WM3064 cells and subsequently conjugated overnight into C. crescentus NA1000 ΔsapA::Pxyl-mkate2 (MFm126) on a PYE agar plate containing 300n μM DAP. The culture was then plated on PYE with 25 μg/ml kanamycin to select for integration of the plasmid and removal of E. coli cells. Successful integrants were incubated in liquid PYE media overnight and plated on PYE supplemented with 3% w/v sucrose to select for excision of the plasmid and sacB gene, leaving the target sequence in the genome. Integration of the sequences was confirmed by colony PCR (with primers SMCAF070 and SMCAF065) using a touchdown thermocycling protocol with an annealing temperature ranging from 72–62 °C, decreasing 1 °C per cycle. The PCR amplicons have been fully sequenced.The BUD-ELM strains were generated using the two-step recombination techniqueSMCAF142: TGGGTCTCAAGGGCGGTTCGGGAGGAGGCSMCAF143: TGGGTCTCAGCAATCCAAACGAGAGTCTAATAGAATGAGGTCSMCAF144: TGGGTCTCCTTGCAACTGGTCTATTTTCCTCTTTTGSMCAF145: TAGGTCTCCCCCTTGTCATCGTCGTCCTTG.SMCAF168: CAGGTCTCTTGTCGCACCTGATTGCCCGASMCAF169: CAGGTCTCTGCTGGGACACCACCGCCAGGSMCAF170: CTGGTCTCCCAGCTGACCCGGCCTTCGGCSMCAF171: CTGGTCTCTGACAATCTATCGATTGTATGGGAAGCCCG.SMCAF070: TATGACGTTTGCTCTATAGCCATCSMCAF065: GAGGATCAGACGTTCGCTTAG.CCAATGATCGTAATACGACTCACTAGTGGGGCCCGCGCCACTCGGTCGCAGGGGGTGTGGGATTTTTTTTGGGAGACAATCCTCATGGCCTATACGACGGCCCAGTTGGTGACTGCGTACACCAACGCCAACCTCGGCAAGGCGCCTGACGCCGCCACCACGCTGACGCTCGACGCGTACGCGACTCAAACCCAGACGGGCGGCCTCTCGGACGCCGCTGCGCTGACCAACACCCTGAAGCTGGTCAACAGCACGACGGCTGTTGCCATCCAGACCTACCAGTTCTTCACCGGCGTTGCCCCGTCGGCCGCTGGTCTGGACTTCCTGGTCGACTCGACCACCAACACCAACGACCTGAACGACGCGTACTACTCGAAGTTCGCTCAGGAAAACCGCTTCATCAACTTCTCGATCAACCTGGCCACGGGCGCCGGCGCCGGCGCGACGGCTTTCGCCGCCGCCTACACGGGCGTTTCGTACGCCCAGACGGTCGCCACCGCCTATGACAAGATCATCGGCAACGCCGTCGCGACCGCCGCTGGCGTCGACGTCGCGGCCGCCGTGGCTTTCCTGAGCCGCCAGGCCAACATCGACTACCTGACCGCCTTCGTGCGCGCCAACACGCCGTTCACGGCCGCTGCCGACATCGATCTGGCCGTCAAGGCCGCCCTGATCGGCACCATCCTGAACGCCGCCACGGTGTCGGGCATCGGTGGTTACGCGACCGCCACGGCCGCGATGATCAACGACCTGTCGGACGGCGCCCTGTCGACCGACAACGCGGCTGGCGTGAACCTGTTCACCGCCTATCCGTCGTCGGGCGTGTCGGGTTCGGGCGGTTCGGGAGGAGGCTCGGGTGACTACAAGGACGACGATGACAAGGGAGTTGGCGTCCCAGGAGTTGGAGTCCCAGGAGGGGGCGTTCCGGGCGCAGGAGTTCCTGGAGTAGGAGTTCCAGGAGTGGGCGTGCCAGGGGTGGGCGTCCCAGGTGGGGGAGTTCCCGGAGCAGGTGTGCCTGGGGGCGGCGTGCCTGGAGTCGGAGTTCCGGGGGTGGGTGTACCGGGTGGAGGCGTACCAGGCGCGGGAGTGCCGGGCGTGGGCGTGCCAGGCGTCGGTGTACCCGGCGTTGGTGTTCCGGGCGGAGGTGTCCCCGGAGCTGGGGTTCCCGGTGGGGGTGTACCGGGCGTCGGGGTTCCCGGTGTGGGTGTCCCAGGTGGCGGCGTTCCCGGGGCGGGCGTACCTGGAGTGGGTGTGCCAGGAGTCGGCGTCCCAGGAGTCGGCGTACCAGGAGGTGGTGTTCCCGGGGCCGGAGTTCCCGGCGGAGGAGTTCCCGGCGTCGGCGTCCCTGGGGTCGGCGTCCCGGGAGGTGGAGTACCCGGAGCAGGAGTGCCGGGAGTCGGTGTACCTGGTGTCGGTGTCCCTGGTGTAGGTGTCCCGGGTGGTGGGGTGCCAGGTGCTGGCGTACCTGGGGGGGGGGTTCCTGGCGTAGGCGTTCCGGGGGTGGGCGTTCCGGGCGGCGGGGTGCCGGGAGCAGGTGTCCCCGGCGTTGGTGTACCGGGGGTTGGTGTCCCAGGCGTAGGTGTGCCCGGTGGAGGGGTGCCGGGAGCTGGAGTGCCTGGAGGGGGTGTACCAGGGGTCGGTGTTCCCGGTGTAGGAGTACCGGGGGGCGGAGTCCCAGGAGCCGGCGTGCCGGGTGTTGGAGTCCCGGGAGTCGGAGTCCCTGGGGTAGGCGTTCCAGGGGGAGGGGTCCCCGGTGCAGGGGTTCCTGGCGGTGGTGTCCCAGGCGGTTCGGGAGGAGGCTCGGGTGCGCATATCGTAATGGTCGATGCATACAAGCCCACGAAAGGAGGTTCAGGCGGCGGAAGCGGTGGTGGAAGCGGAGGTGGGTCAGGCGGAGGCTCAGGGGGAGGTTCGGGTGGCGGTTCGGGAGGAGGCTCGGGTGCTGACCCGGCCTTCGGCGGCTTCGAAACCCTCCGCGTCGCTGGCGCGGCGGCTCAAGGCTCGCACAACGCCAACGGCTTCACGGCTCTGCAACTGGGCGCGACGGCGGGTGCGACGACCTTCACCAACGTTGCGGTGAATGTCGGCCTGACCGTTCTGGCGGCTCCGACCGGTACGACGACCGTGACCCTGGCCAACGCCACGGGCACCTCGGACGTGTTCAACCTGACCCTGTCGTCCTCGGCCGCTCTGGCCGCTGGTACGGTTGCGCTGGCTGGCGTCGAGACGGTGAACATCGCCGCCACCGACACCAACACGACCGCTCACGTCGACACGCTGACGCTGCAAGCCACCTCGGCCAAGTCGATCGTGGTGACGGGCAACGCCGGTCTGAACCTGACCAACACCGGCAACACGGCTGTCACCAGCTTCGACGCCAGCGCCGTCACCGGCACGGGCTCGGCTGTGACCTTCGTGTCGGCCAACACCACGGTGGGTGAAGTCGTCACGATCCGCGGCGGCGCTGGCGCCGACTCGCTGACCGGTTCGGCCACCGCCAATGACACCATCATCGGTGGCGCTGGCGCTGACACCCTGGTCTACACCGGCGGTACGGACACCTTCACGGGTGGCACGGGCGCGGATATCTTCGATATCAACGCTATCGGCACCTCGACCGCTTTCGTGACGATCACCGACGCCGCTGTCGGCGACAAGCTCGACCTCGTCGGCATCTCGACGAACGGCGCTATCGCTGACGGCGCCTTCGGCGCTGCGGTCACCCTGGGCGCTGCTGCGACGCTAGCTGACTGGGAAAACCCTGGCGTTAATCGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTC.C. crescentus strains into 80 mL of PYE in a 250 mL glass flask. All cultures were grown in an Innova 44 incubator shaker with a 2-inch orbit. These cultures were grown at 30 °C at 250 rpm, and BUD-ELMs typically formed within ~24–30 h. To explore the effect of growth parameters on BUD-ELM size, the flask volume, shaking speed, and culture volume were varied from 125 to 500 mL, 0 to 250 rpm, and 25 to 160 mL, respectively. The complete list of conditions tested can be found in Supplementary Tables Unless indicated otherwise, BUD-ELMs were grown by inoculating a single colony of 2 piece was broken off from the desiccated material and inoculated into 80 mL of fresh PYE and grown under standard conditions. We detected BUD-ELM formation in 48 h for material dried over 7 or 14 days, and in 72 h for material dried over 21 days.To test the ability of BUD-ELMs to re-seed their own growth, BUD-ELMs grown under standard conditions were collected, transferred in a petri dish for 7, 14, or 21 days and left on the bench at room temperature. The material dried out in 24–48 h. To re-seed material growth, a ~0.3 to 0.5 cmE. coli were assembled from existing constructs  by substituting the mRFP sequence with the GFP sequence (below). Similarly, plasmids pSMCAF032 and pSMCAF029 used for the expression of GDH and SpyCatcher-GSH from E. coli were assembled by introducing the GDH sequence in the same position. These plasmids were transformed into chemically competent BL21(DE3) cells (New England Biolabs – C2527H); single transformants were selected using ampicillin resistance.Plasmids pSMCAF015 and pSMCAF016 used for the expression of GFP and SpyCatcher-GFP from ATGCGTAAAGGCGAAGAGCTGTTCACTGGTGTCGTCCCTATTCTGGTGGAACTGGATGGTGATGTCAACGGTCATAAGTTTTCCGTGCGTGGCGAGGGTGAAGGTGACGCAACTAATGGTAAACTGACGCTGAAGTTCATCTGTACTACTGGTAAACTGCCGGTTCCTTGGCCGACTCTGGTAACGACGCTGACTTATGGTGTTCAGTGCTTTGCTCGTTATCCGGACCATATGAAGCAGCATGACTTCTTCAAGTCCGCCATGCCGGAAGGCTATGTGCAGGAACGCACGATTTCCTTTAAGGATGACGGCACGTACAAAACGCGTGCGGAAGTGAAATTTGAAGGCGATACCCTGGTAAACCGCATTGAGCTGAAAGGCATTGACTTTAAAGAAGACGGCAATATCCTGGGCCATAAGCTGGAATACAATTTTAACAGCCACAATGTTTACATCACCGCCGATAAACAAAAAAATGGCATTAAAGCGAATTTTAAAATTCGCCACAACGTGGAGGATGGCAGCGTGCAGCTGGCTGATCACTACCAGCAAAACACTCCAATCGGTGATGGTCCTGTTCTGCTGCCAGACAATCACTATCTGAGCACGCAAAGCGTTCTGTCTAAAGATCCGAACGAGAAACGCGATCATATGGTTCTGCTGGAGTTCGTAACCGCAGCGGGCATCACGCATGGTATGGATGAACTGTACAAATAA.GACGTTCCGCTGACCCCGAGCCAGTTTGCGAAAGCGAAAAGCGAGAACTTCGACAAAAAAGTCATCCTGAGCAACCTGAATAAACCGCACGCTCTGCTGTGGGGTCCGGATAATCAGATTTGGCTGACCGAACGCGCAACCGGTAAAATTCTGCGCGTTAACCCGGAAAGCGGCAGCGTTAAAACCGTCTTTCAGGTTCCGGAAATCGTTAACGACGCAGACGGTCAAAACGGTCTGCTGGGTTTTGCGTTTCATCCGGACTTCAAAAACAACCCGTACATCTACATCAGCGGCACCTTCAAAAACCCGAAAAGTACCGACAAAGAGCTGCCGAATCAGACCATCATCCGTCGCTATACCTACAACAAAAGCACCGACACCCTGGAAAAACCGGTTGATCTGCTGGCAGGTCTGCCGAGTAGTAAAGATCATCAGAGCGGTCGTCTGGTAATTGGTCCGGACCAGAAAATCTACTATACCATTGGCGATCAGGGCCGTAACCAACTGGCATACCTGTTTCTGCCGAACCAAGCACAACATACCCCGACCCAACAAGAACTGAACGGCAAAGACTACCACACCTACATGGGCAAAGTTCTGCGTCTGAATCTGGACGGTAGCATTCCGAAAGACAACCCGAGCTTCAACGGCGTTGTTAGCCATATCTATACCCTGGGTCACCGTAATCCGCAAGGTCTGGCATTTACCCCGAACGGTAAACTGCTGCAGTCTGAACAGGGTCCGAATTCTGACGACGAAATCAACCTGATCGTTAAAGGCGGCAATTACGGTTGGCCGAACGTTGCAGGCTATAAAGACGATAGCGGCTATGCATACGCGAATTATAGCGCAGCGGCAAACAAAAGCATCAAAGACCTGGCCCAGAACGGTGTTAAAGTTGCAGCAGGCGTTCCGGTTACCAAAGAAAGCGAGTGGACCGGCAAAAACTTTGTTCCGCCGCTGAAAACCCTGTATACCGTCCAGGACACCTACAACTATAACGATCCGACCTGCGGCGAAATGACCTATATTTGCTGGCCGACCGTTGCACCGAGTTCTGCATACGTTTACAAAGGCGGCAAAAAAGCGATCACCGGTTGGGAAAATACCCTGCTGGTTCCGAGTCTGAAACGCGGCGTTATCTTCCGCATCAAACTGGATCCGACCTATAGTACCACCTACGACGATGCCGTTCCGATGTTCAAAAGCAACAACCGTTATCGCGACGTTATTGCAAGTCCGGACGGTAACGTTCTGTACGTTCTGACCGATACCGCAGGTAACGTTCAGAAAGACGACGGTAGCGTTACCAATACCCTGGAAAATCCGGGTAGCCTGATCAAATTCACCTACAAAGCGAAATGA.E. coli BL21(DE3) harboring plasmids pSMCAF015 and pSMCAF016, for expression of GFP and SpyCatcher-GFP, respectively, were inoculated in 25 mL of RM minimal media with 0.2% w/v glucose and 100 µg/mL ampicillin. After ~16 h of growth at 37 °C and 250 rpm, cells were used to inoculate 0.5 L of RM minimal media with 0.2% v/v glycerol, 100 µg/mL ampicillin and 0.0004% antifoam (Antifoam 204) to a final OD600 ~0.05. The cultures were allowed to grow at 37 °C until mid-log phase. Protein production was induced with 0.2% w/v l-arabinose with incubation at 30 °C for ~17 h.Single colonies of g for 30 min, resuspended in lysis buffer  and lysed using Avestin Emulsiflex C3 Homogenizer. The lysate was centrifuged at 12,000 × g for 1 h and the supernatant was collected for protein purification. The proteins were purified using Immobilized Metal Affinity Chromatography (IMAC) with a HisTrap FF column and buffers containing 50 mM Tris pH 8.0, 300 mM NaCl, 5% v/v glycerol, and 10–250 mM Imidazole. After protein purity was confirmed by SDS-PAGE, the protein was dialyzed into TEV-cleavage buffer  and the 6 x His-tag was cleaved using TEV protease by agitation at 4 °C for 4 h. The cleaved protein was stored at −80 °C in 50 mM NaPO4 pH 8.0, 300 mM NaCl, and 5% v/v glycerol.Cells were harvested by centrifugation at 8000 × 2O and lyophilized for 5 h in a Labconco Freezone 4.5 freeze dryer. Tubes were then weighted.Eight BUD-ELMs were grown under standard conditions. Samples were harvested from liquid cultures, placed in Eppendorf tubes, washed once in ddHl-lysine coated silicon substrates were immersed in Falcon™ round-bottom polypropylene culturing tubes containing 3 mL fresh C. crescentus cell culture at an OD600 of 0.3–0.5. Culture tubes were then centrifuged at 3000 × g for 10 min to immobilize the cells onto the silicon substrate. The silicon substrate was washed with 2 mL of sterile PYE to remove loosely-bound cells before being mounted to a metal puck and transferred to the AFM sample stage. In situ AFM was performed on an Asylum Cypher AFM using soft tapping mode. A fluid cell and two syringe pumps were assembled to control liquid flow and PYE medium was supplied to maintain cell viability during imaging. The AFM probe consisted of a sharp silicon tip on a silicon nitride cantilever  with a spring constant of 0.09 N/m. Cells were imaged in native state without fixation. A 100–200 mV amplitude setpoint was used to apply minimum forces (~0.2 nN) to cells during imaging.Poly-2 precleaned silicon substrate was dipped into the pellicle forming cell culture with an entry angle of ~60° perpendicular to the water surface. The silicon substrate was then retrieved, and the pellicle structure was dried under an N2 atmosphere for 2 h. The dried pellicle structure on the substrate was mounted to a metal puck and transferred to the AFM sample stage. AFM imaging was performed on an Asylum Cypher AFM using soft tapping mode in air. A Tap-150 tip (BudgetSensors) with a 5 N/m force constant was used to image the pellicle structure.To bind the BUD-ELM pellicle to a silicon substrate, a 2 cmSmall pieces of BUD-ELMs grown under standard conditions were placed between a slab of PYE agarose (1.5% w/v) and a glass coverslip-bottomed 50-mm Petri dish with a glass diameter of 30 mm (MatTek Corporation) and imaged with an optical inverted microscope. Optical microscopy data were acquired using the software NIS-Elements AR (version 4.51.01). All microscopy pictures presented were generated using ImageJ software (version 2.0.0-rc-69/1.52p).d-xylose—to induce the expression of mKate2, in a 125 mL flask and grown for 24 h at 30 °C at a shaking speed of 250 rpm. BUD-ELMs of similar dimensions were collected and washed twice with 1 mL of 0.01 M Phosphate-buffered saline (PBS), in a centrifuge tube. They were then incubated in 1 mL of 0.01 M PBS, at 30 °C, with the following staining agent: 80 µg of SpyCatcher-GFP or GFP for 1 h, 1% Congo Red (Thermo Fisher Scientific—D275) or 100 µg DiO (DiOC18(3) − 3,3′-Dioctadecyloxacarbocyanine Perchlorate) for 20 min. Samples were washed three times with 1 mL 0.01 M PBS and then a small amount was placed between a slab of PYE agarose (1.5% w/v) and a glass coverslip-bottomed 50-mm Petri dish with a glass diameter of 30 mm (MatTek Corporation). To acquire the low-magnification images  to distinguish the matrix from the cells.Single colonies of BUD-ELM strain RCC002) were inoculated in 30 mL PYE with 0.15%  were inoC. crescentus BUD-ELM strain (RCC002) were cultured in standard (shaking) or static (not shaking) conditions until they reached stationary phase . The supernatant of each culture was extracted and loaded onto a TGX Stain-Free™ gel (Biorad). After running, the gel was transferred to a 0.2 μm nitrocellulose membrane and blocked for 1 h at room temperature with SuperBlock™ blocking buffer (Thermo Scientific). Membranes were then washed four times in TBST buffer before incubation in a 1:5000 dilution of Monoclonal ANTI-FLAG® antibody  antibody from Sigma Aldrich – A8592-.2MG) produced in mouse, clone M2, purified immunoglobulin, buffered aqueous glycerol solution) solution for 1 h at room temperature. Membranes were washed an additional four times in TBST buffer before Clarity Max Western ECL Substrate (Biorad) was applied and the membrane was imaged for chemiluminescence. Each sample lane represents an independent sample, grown from separate individual colonies.For immunoblot analysis of culture supernatant, cultures of ΔELP60 BUD-ELM strains were grown under standard conditions. Planktonic cells from each colony were collected and washed 3 times in fresh PYE media to remove any free BUD protein not attached to the cell surface. After washes, all samples were normalized to an OD600 value of 0.225 in 0.5 x PYE, 1 x Laemmli buffer. Each sample was then boiled for 5 min, and 10 uL was loaded onto a TGX Stain-Free™ gel (Biorad). All subsequent steps follow the protocol for immunoblotting described above. Protein molecular weight has been determined by image analysis with ImageJ.For whole-cell immunoblot analysis, the original and L) values.To determine the apparent BUD-ELM size, the bottom of glass flasks where BUD-ELM grew was imaged using a Canon EOS 77D camera. Flasks were positioned within a reflective photobox on a clear plastic surface such that the bottom of the flask stood approximately 11.5 cm above the camera lens. These images were separated into RGB channels using MATLAB R2020b. A subset of the blue channel images was then input into the image classification software ilastik (version 1.3.3), as a training set for the autocontext workflow. The first stage of training separated images into three different classifications: background, scattered material, and bundled material. Scattered material was defined as overlapping regions of small aggregates not associated with each other, whereas bundled material referred to larger, connected pieces of material. The second stage of training distinguished bundled material from the rest of the image. Both stages of training utilized all 37 features provided within the ilastik workflow . From the results of the training set, the second stage segmentation masks for all blue channel images were calculated, and loaded into MATLAB R202b. From these masks, the flat area of each piece of material was calculated, and the top five percentile of size from each image was averaged to yield a representative size measurement. For each image, a conversion rate between pixels and squared centimeters was determined using the standard flask diameter as a reference point. Size measurements were averaged between samples and plotted with respect to their calculated (d5)(kLa); Ne’ – modified Newton number (dimensionless); P – power input (W); Re – Reynold’s number (dimensionless); VL – culture volume (m3); ηapp – dynamic apparent viscosity (Pa∙s); ρ – liquid density (kg/m3); d0 – orbital shaking diameter (m); D – diffusion coefficient (m2/s); v – kinematic viscosity (m2/s); g – acceleration of gravity (m/s2).ELP60, and ∆rsaA1–250 strains, respectively) were evaluated on a strain-controlled rheometer (ARIES G2) equipped with an 0.1 rad 8-mm diameter cone plate. BUD-ELMs were grown in standard conditions. An approximate volume of 100–200 µL of BUD-ELMs were collected into a 1.5 mL centrifuge tube and spun for 10 s at 3200 rcf with a mini centrifuge (VWR® – C0803). This allowed for the material to collect at the bottom as a homogeneous paste. The supernatant was removed and 150uL of fresh PYE were added on top of BUD-ELMs to prevent desiccation. Strain sweep experiments from 0.1 to 100% strain amplitudes were performed at a fixed frequency of 3.14 rad/s. Frequency sweep experiments from 100 to 0.1 rad/s were performed at a 0.35% strain amplitude. Data were acquired using TRIOS software (version 4.2.1.36612).The rheological properties of BUD-ELMs produced from strains RCC002, RCC004 and MFm152  and incubated with 7 mL of 6 ppm CdCl2 (Sigma Aldrich – 202908) in ddH2O for 90 min on an orbital shaker. After incubation, the Cd2+ concentration of the supernatant was measured by ICP-MS. Specifically, 5 µL of the supernatant was diluted in 4.995 mL 1% HNO3 with 5 µg/mL Indium (In), as standard for data analysis (Perkin Elmer N9303741). This diluted solution was run on a Perkin Elmer Nexion 300 ICP-MS with two isotopic measurements (Cd2+ 111 and Cd2+ 112) and In 115 as the internal standard. Data was acquired using Syngistix software.To measure the ability of BUD-ELMs to bind CdE. coli BL21(DE3) harboring pSMCAF032 and pSMCAF029, for expression of GDH and SpyCatcher-GDH, respectively, were inoculated in 25 mL of Terrific broth (TB) with 100 µg/mL ampicillin. After ~16 h of growth at 37 °C and 250 rpm, cells were used to inoculate 0.5 L of TB with 0.02% antifoam (Antifoam 204) and 100 µg/mL ampicillin to a final OD600 ~0.05. The cultures were allowed to grow at 37 °C until the mid-log phase. Protein production was induced with 0.2% w/v L-arabinose with incubation at 30 °C for ~17 h.Single colonies of g for 5 min, resuspended in lysis buffer (0.01 M MOPS) and lysed using sonication. After centrifugation at 12,000 × g for 1 h, the GDH (or SpyCatcher-GDH) in the supernatant was reconstituted by adding a final concentration of 3 mM Ca2+ and 0.06 mM PQQ and incubated for 15 min at 4 °C. BUD-ELMs, washed once with 0.01 M PBS, were incubated with reconstituted and non-reconstituted cell lysates for 2 h at 4 °C and then washed three times with 0.01 M PBS. A small piece of functionalized BUD-ELMs , 1 mL DCPIP (20 mg dissolved in 5 mL of DI water), and 1 mL phenazine methosulfate (PMS) (45 mg dissolved in 5 mL of DI water) were prepared. Analytical samples (~3 uL) were mixed with the reagent to 190 µL. The reaction was initialized by adding 10 µL glucose (2 M). Glucose consumption was correlated to the consumption of DCPIP (2:1 ratio), which was quantified colorimetrically by absorption at 600 nm. A representative curve is shown in Supplementary Fig. The activity of GDH functionalized material was quantified with a modified colorimetric 2,6-dichlorophenol (DCPIP) assayFigure Figures Figures The blots shown in Fig. Further information on research design is available in the Supplementary InformationReporting SummaryPeer Review File


Liquid–liquid phase separation (LLPS) governs a variety of mesoscale cellular processes. However, less is known about how cells utilize LLPS to drive cellular function. Here, we examined the destruction complex (DC), an organelle which controls Wnt signaling and whose components phase separate. Through a combination of advanced microscopy, CRISPR, computational modeling, and optogenetics, we find that the DC is nucleated by the centrosome and that this nucleation drives efficient signal transduction. Our work not only uncovers a biological function for LLPS but also highlights nucleation as a general method for controlling the function of intracellular condensates. Finally, our findings suggest a thermodynamic coupling between Wnt signal transduction and the cell cycle which could lead to insights into Wnt-driven cancers. Wnt signal transduction is controlled by the destruction complex (DC), a condensate comprising scaffold proteins and kinases that regulate β-catenin stability. Overexpressed DC scaffolds undergo liquid–liquid phase separation (LLPS), but DC mesoscale organization at endogenous expression levels and its role in β-catenin processing were previously unknown. Here, we find that DC LLPS is nucleated by the centrosome. Through a combination of CRISPR-engineered custom fluorescent tags, finite element simulations, and optogenetic tools that allow for manipulation of DC concentration and multivalency, we find that centrosomal nucleation drives processing of β-catenin by colocalizing DC components to a single reaction crucible. Enriching GSK3β partitioning on the centrosome controls β-catenin processing and prevents Wnt-driven embryonic stem cell differentiation to mesoderm. Our findings demonstrate the role of nucleators in controlling biomolecular condensates and suggest tight integration between Wnt signal transduction and the cell cycle. The canonical Wnt signaling pathway is a conserved , morphog2Extracellular Wnt ligands inhibit DC function through a mechanism that is still unclear, but likely involves selective recruitment of DC components to the signalosome, a biomolecular condensate on the plasma membrane that includes Wnt/Frizzled/LRP5/6 clusters  and, whe8The “molecular crucible” model posits that DC LLPS promotes β-cat degradation in Wnt OFF conditions  via concA biophysical mechanism for regulating condensates is control over their nucleation. This principle was recently explored with a synthetic optogenetic system , but theSlimb  and localization of β-cat at the cell membrane, consistent with previous work in fixed specimens  (Movies S2 and S3). β-cat puncta became more difficult to distinguish at higher cytoplasmic concentrations produced by activated Opto-LRP6, but dissolution nearly always preceded appreciable dilute-phase β-cat accumulation, indicating that they were not simply obscured by higher background levels. Further, of light-stimulated cells, those that were resistant to optogenetic activation maintained their β-cat puncta . Together, these results indicate that activation of the Wnt pathway causes perinuclear puncta to dissolve, and the presence of these puncta is inversely related to Wnt pathway activation at the population and single-cell levels.To establish whether directly activating the Wnt receptor controls the existence of the puncta, we transduced to-LRP6) K and L. to-LRP6) F. We fout puncta I and J. CSNK1A1, GSK3B, and AXIN1, genes encoding the kinases CK1α and GSK3β that sequentially phosphorylate β-cat in the DC, and the primary DC scaffold.We next sought to determine 1) what, if any, cellular structure was organizing these puncta, 2) whether all DC components were colocalized with puncta, and 3) whether these were solid or liquid-like condensates. Because of the sensitivity of LLPS systems to protein concentration , we deciSI Appendix, Fig. S2A): We observed single condensates in G1, two condensates in G2/S, and a “finger-like” pattern—suggesting association with the mitotic spindle—during late mitosis. These observations, combined with previous reports of perinuclear enrichment of CK1α, GSK3β, and Axin1 in fixed cells  confirmed that tdmRuby3-CK1α, tdmRuby3-GSK3β, and tdmRuby3-β-cat puncta were indeed colocalized to the centrosome.We found that all tagged proteins were localized into one or two perinuclear puncta A. Timelaed cells –31, led ed cells B and GM1Left) that colocalized with centrosomal markers  and replicated following cell cycle progression . As protein concentration increased, Axin1, but not APC, caused formation of extracentrosomal puncta throughout the cytoplasm . Interestingly, extracentrosomal Axin1 condensates did not reliably induce formation of extracentrosomal GSK3β condensates in these experiments, but often resulted in deenrichment of centrosomal puncta . We reason that this was due to extracentrosomal Axin1 condensates competing for relatively scarce GSK3β, thereby diluting across all condensates in the cytoplasm.When overexpressed, Axin and APC cross the phase boundary and form liquid condensates in the cytoplasm that are hypothesized to concentrate DC kinases and β-cat . The facytoplasm C–E. To dSI Appendix, Fig. S2F). These findings establish that all DC components necessary for phosphorylating β-cat, prior to its ubiquitination, are localized at the centrosome throughout the cell cycle and suggest that DC centrosomal nucleation is generalizable to multiple cell types.The 293Ts are commonly used in experiments probing DC mesoscale structure in vivo , 10, butNext, we sought to determine the material state of the centrosomal DC using FRAP on CRISPR-tagged CK1α, GSK3β, and β-cat, as well as of Axin1 and APC at low levels of induction. All centrosomal DC components exhibited mean half-maximal recovery times (τ1/2) between 10 s and 60 s E—like inSI Appendix, Fig. S3 A–D). Indeed, synthetic DC scaffolds with these simple attributes have been shown to rescue aberrant Wnt signaling . We found that the nucleated system processed β-cat and its intermediates more quickly . See SI Appendix, Fig. S3F). This efficiency gain was maintained over a large range of reaction rates . As expected, in systems with high reaction rates, the effect of nucleated phase separation is no longer observed.To test the effects of nucleation on β-cat processing, we compared simulations in the presence and absence of a nucleation region C. We fou quickly D over a SI Appendix, Fig. S3 H and I, Movie S6). Together, these results demonstrate that nucleation of DC components has the potential to increase β-cat processing and that a tunable control parameter of this process is the free energy of mixing.Given our findings that nucleation drives efficient processing of β-cat, we hypothesized that In silico analysis of the DC indicates that processing efficiency in the presence of a nucleator is dependent on client condensation. Imaging of GSK3β showed relatively weak enrichment in centrosomal puncta compared to CK1α, suggesting that increasing nucleation of GSK3β would increase the degradation rate of β-cat in vivo. Changing concentration alters both propensity to undergo LLPS and reaction rate  and therMovie S7). Notably, activation of Opto-GSK3 strictly resulted in the formation of one or two perinuclear puncta and did not form extracentrosomal condensates, contrasting with results from studies using Cry2 alone  and eGFP to human GSK3β (“Opto-GSK3” hereafter) and stably transduced it into 293Ts A. Upon ly2 alone . Thus, wSI Appendix, Fig. S4 A and B). We observed a similar effect when analyzing total β-cat by Western blotting and immunofluorescence staining . Given the modest accumulation of β-cat in response to Wnt-3a in 293Ts, we tested to see whether Opto-GSK3 clustering was sufficient to blunt β-cat accumulation induced by either CHIR or a Dox-inducible β-cat overexpression construct. Indeed, activation of Opto-GSK3 also inhibited both methods for driving β-cat accumulation in a light-dependent manner . These results demonstrate that increasing DC client nucleation at the centrosome dictates Wnt signal transmission across a wide range of activation regimes.To determine whether increased centrosomal condensation of GSK3β controlled Wnt signal transmission, we activated Opto-GSK3 in cell lines with three distinct methods for increasing the cellular concentration of β-cat: ligand-induced, kinase inhibition, and dox-induced gene up-regulation. We found that Opto-GSK activation abolished both Wnt-3a-induced β-cat accumulation and transcriptional activation as measured by TOPFlash fluorescence D and E. Changes in β-cat concentration differentiate a variety of stem cell populations, including human embryonic stem cells (hESCs) , 40. HavBuilding on recent discoveries suggesting that LLPS plays a role in DC structure, we sought to understand how the biophysics of DC proteins regulate DC function in live cells. Through a combination of superresolution microscopy, in silico modeling, and optogenetic methods to isolate and probe the phase diagram, we discovered that the mesoscale structure of the DC is a liquid condensate nucleated by the centrosome. The complementarity of these methods allowed us to identify a function for nucleation: acceleration of the catalytic action of DC proteins, thereby promoting efficient processing of β-cat.The presence of many cytoplasmic Axin1 and APC droplets in mildly overexpressed cellular conditions  has beenOur results support a “molecular crucible” model of β-cat degradation, in which multivalent DC scaffolds concentrate DC clients in nucleated droplets to increase β-cat phosphorylation rate. Assembly line models for β-cat degradation have been proposed , 42, andWe found that centrosomal DCs cease to concentrate β-cat under Wnt ON and GSK3β chemical inhibition, but the mechanism for this change remains unclear. Multiple DC components that bind β-cat—including Axin1, GSK3β, and CK1α—are also binding partners of the Frizzled-LRP6 signalosome , 45, a kDrosophila embryos leads to only minor tissue-level defects in canonical Wnt signaling and overall morphology  coupling the DC to the centrosome? Axin1 is known to associate with γ-tubulin  and is arphology , 48, indFinally, centrosomal nucleation of the DC suggests a potential function in coordinating cell cycle progression with Wnt signaling. We found two DC droplets in cells with duplicated centrosomes, suggesting that the DC is split along with centrosomes during mitosis. Nonnucleated droplets would be randomly partitioned into daughter cells, leading to potentially detrimental asymmetry in Wnt signaling capacity of the growing tissue. Cell cycle synchronization could be a method of reducing heterogeneity in Wnt-induced stem cell differentiations.Overall, our studies suggest an integral role for LLPS nucleation in regulating the activity of membraneless organelles in vivo. The power of observing proteins in their endogenous contexts, coupled with the ability to precisely tune interaction strength without altering protein function or concentration, enables the functional dissection of membraneless organelles.2 Dulbecco’s Modified Eagle Medium, high glucose GlutaMAX  medium supplemented with 10% fetal bovine serum , and 1% penicillin-streptomycin. The hiPSC WTC was gifted by the B.P. laboratory (purchased from Coriell). The hiPSCs were propagated on Matrigel-coated tissue culture plates using serum-free essential 8 (Gibco) culture conditions in standard environments consisting of 5% carbon dioxide at 37 °C. Experiments in hESC lines were performed using the H9 hESC cell line purchased from the William K. Bowes Center for Stem Cell Biology and Engineering at University of California, Santa Barbara (UCSB). Cells were grown in mTeSR Plus medium (Stem Cell Technologies) on Matrigel (Corning)-coated tissue culture dishes and tested for mycoplasma in 2-mo intervals.Human 293T cells were cultured at 37 °C and 5% COPiggyBac (CMV) Backbone fwd: tgacgcccgccccac rev: ggtaagctttttgcaaaagcctaggcc. H2B + 18AA linker fwd: cctaggcttttgcaaaaagcttaccatgccagagccagcgaagtc rev: GCATATTTTCCTTGATGAGTTCACTCATccCagTatGtcCgcCggAg. mTagBFP2 fwd: ATGAGTGAACTCATCAAGGAAAATATGCACATG rev: CGTCCCCGCAGGTCAACAAACTTCCGCGACCTTCTCCGCTCCCATTGAGCTTATGGCCGAGTTTGCTG.-3′X-Flag-NLS-Cas9-HA-Avidin fwd: GGAAGTTTGTTGACCTGCGGGGACGTGGAAGAAAACCCGGGTCCAgactataaggaccacgacggagactac rev: gctgcgggtcgtggggcgggcgtcaggatccagacgccgcagThe pPig_H2B-mTagBFP2::t2A::Cas9-Avidin was constructed via subcloning human H2B, mTagBFP2, and Cas9-Avidin provided by M.Z.W. into an expression vector bearing a cytomegalovirus CMV promoter and flanking PiggyBac transposase-compatible inverted terminal repeats using Gibson Assembly  according to supplier instructions. Each of the PCR fragments used was amplified using the following primers:rd gen tet ON-responsive promoter, and EF1α-driven Blasticidin selection cassette. The following primers were used: XLone Backbone fwd: taaactagtagaccacctcccctgcg, rev: ggtacctttacgagggtaggaagtgg, human Axin1 fwd: cacttcctaccctcgtaaaggtaccatgaatatccaagagcagggtttcccc, rev: CCATgctTCCgCCgCCACTACCgCCgtccaccttctccactttgccgatgatc, 7AA link- tdmRuby3 fwd: GGcGGTAGTGGcGGcGGAagcATGGTTAGCAAAGGGGAGGAGC, rev: gcaggggaggtggtctactagtttaCTTGTACAGCTCGTCCATGCCGXLone-Axin-tdmRuby3 was constructed via PCR and Gibson Assembly, subcloning from the following constructs: Flag-Axin1 purchased from Addgene (#109370), tdmRuby3 from M.Z.W. into XLone-GFP purchased from Addgene (#96930) containing-3′XLone-bCat-tdmRuby3 was constructed via PCR and Gibson Assembly, subcloning from the following constructs: XLone-Axin-tdmRuby3 (above) and Human Beta-catenin GFP purchased from Addgene (#71367). The following primers were used: XLone Backbone fwd: GGcGGTAGTGGcGGcGGAagcATGGTTAGCAAAGGGGAGGAGC, rev: ggtacctttacgagggtaggaagtgg, human bcat fwd: cacttcctaccctcgtaaaggtaccatggctactcaagctgatttgatggagttg, rev: CCATgctTCCgCCgCCACTACCgCCcaggtcagtatcaaaccaggccagcpPig-Hygro Backbone fwd: GGACGTGGAAGAAAACCCGGGTCCAatgggtaaaaagcctgaactcaccgc, rev: cattccacagggtcgacagtacaagc,Cuo + CMV fwd: cttgtactgtcgaccctgtggaatgcgttacataacttacggtaaatggcccgc,rev: actgatcatatgaagctgcagccatgaattcggtaccggatccagtcgactag,APC fwd: atggctgcagcttcatatgatcagttgttaaagcaag,rev: CCATgctTCCgCCgCCACTACCgCCaacagatgtcacaaggtaagacccagaatg,7AAlinker-tdmirfp670nano fwd: GGcGGTAGTGGcGGc,rev: ggcgccaaaacccggcgcggaggccttaGGACTGCTGTATTGCAATGCCAACTAC,UbC-CymR-V5-T2A fwd: ggcctccgcgccggg,rev: TGGACCCGGGTTTTCTTCCACGTCCCCGCAGGTCAACAAACTTCCGCGACCTTCTCCGCTCCCcgtagaatcgagaccgaggagaggThe pPig_CuO-APC-tdmIRFP670::CymR was constructed via PCR and Gibson Assembly from the following constructs: pCuo CA Rac1 CMV + cumate operon purchased from Addgene (#84643), human APC open reading frame (ORF) purchased from Addgene (#16507), tdmirfp670nano from M.Z.W., and human ubiquitin C-driven CymR Cuo repressor purchased from Addgene (#119907) into pPig-Hygro transposase backbone from M.Z.W. PCR fragments were amplified using the following primers:Arabadopsis thaliana, tdmIRFP from M.Z.W. and human GSK3β purchased from Addgene (#16260) ORFs were supplied to VectorBuilder for cloning and EF1α-driven expression into third-generation lentiviral backbone. Vectorbuilder provided the desired final, sequenced plasmid.The pLV_Cry2-tdeGFP-GSK3b was obtained via synthesis and cloning services provided by Vector Builder Inc. Full details are available upon request, but, briefly: primary plasmids containing ATGGCTGAAGGCAGCGTGGCCCGACAGCCAGACCTTTTGACTTGTGACGATGAACCAATCCACATACCGGGGGCAATACAACCTCATGGTCTCCTTCTGGCGCTTGCTGCCGACATGACTATAGTGGCCGGCTCTGACAACTTGCCGGAATTGACCGGACTTGCTATTGGGGCGTTGATTGGGCGCTCTGCCGCTGATGTATTTGATTCCGAGACACATAATAGGCTTACTATAGCCCTCGCCGAACCAGGGGCTGCCGTCGGCGCTCCTATAACAGTTGGGTTCACGATGCGAAAAGATGCTGGGTTCATTGGTAGCTGGCATCGCCACGATCAACTTATCTTCCTTGAGCTTGAACCCCCTCAACGGGACGTTGCGGAACCCCAAGCTTTCTTTAGAAGGACCAATTCAGCCATAAGGCGCCTTCAGGCCGCAGAGACATTGGAGTCCGCGTGTGCGGCAGCAGCGCAGGAAGTACGAAAGATCACGGGATTTGACCGGGTTATGATTTACAGATTCGCATCTGATTTCTCCGGGGAAGTCATCGCGGAGGATCGGTGTGCAGAAGTGGAAAGCAAGCTTGGTTTGCATTACCCCGCATCTACGGTTCCGGCCCAAGCGAGGAGACTGTATACGATAAACCCAGTGAGGATCATACCTGACATAAATTATAGACCGGTTCCCGTTACGCCAGACCTGAACCCCGTCACAGGCAGGCCAATAGACTTGTCTTTTGCAATCCTGCGGTCAGTCTCACCTGTTCACCTCGAGTTTATGAGGAACATAGGGATGCATGGGACGATGAGCATCTCAATCCTGAGAGGTGAACGGCTCTGGGGACTTATTGTTTGTCATCATCGCACACCGTATTACGTTGACCTTGATGGTCGCCAGGCCTGCGAACTCGTAGCTCAAGTATTGGCCTGGCAGATCGGTGTTATGGAGGAAAGCGGTCATGGGACTGGGAGTACAGGTAGCGGCAGCTCTAGTGGCACCTCCandTAGCGGCAGCTCTAGTGGCACCTCCATGGCAGAAGGGTCCGTAGCAAGGCAACCTGACTTGTTGACCTGTGATGATGAACCGATTCACATTCCTGGAGCAATTCAACCGCATGGGCTGCTCCTTGCTTTGGCAGCGGACATGACGATCGTCGCCGGCTCCGATAACCTGCCCGAGTTGACGGGCTTGGCGATAGGAGCCCTGATAGGCCGCTCAGCCGCTGACGTATTCGATAGCGAAACGCATAACCGGCTTACAATCGCCTTGGCTGAACCGGGCGCGGCCGTGGGAGCACCGATTACTGTAGGCTTTACAATGAGAAAAGACGCCGGCTTTATCGGGTCATGGCACCGACATGACCAGCTGATTTTCCTGGAATTGGAGCCCCCGCAGCGGGATGTAGCCGAACCACAGGCCTTCTTCCGGCGCACTAACTCCGCAATTAGGAGACTGCAGGCAGCTGAGACTTTGGAATCAGCATGCGCGGCAGCTGCACAAGAAGTCCGGAAAATCACGGGTTTTGACCGAGTCATGATCTATAGATTCGCGAGCGATTTCTCAGGAGAAGTTATTGCGGAAGACCGATGCGCGGAGGTAGAATCTAAGCTTGGGTTGCACTACCCCGCCTCCACCGTTCCGGCGCAAGCCAGACGGCTCTATACCATTAATCCGGTGCGGATCATTCCAGATATAAATTACCGGCCTGTACCTGTGACACCGGATTTGAACCCTGTCACGGGCCGACCGATAGACCTCAGCTTCGCTATATTGCGATCTGTGTCACCGGTCCACCTCGAGTTTATGAGGAATATAGGCATGCATGGTACAATGTCCATTTCCATTCTCCGGGGTGAACGGCTTTGGGGCCTCATCGTTTGTCACCATCGAACACCGTATTACGTCGATCTCGACGGCAGACAGGCATGTGAGTTGGTCGCTCAGGTACTCGCTTGGCAGATAGGGGTAATGGAGGAGThe pPig_8XTOP_tdIRFP_Puro was constructed via PCR and Gibson Assembly from the following constructs: pPig_H2B-mTagBFP2::t2A::Cas9-Avidin (above), M50 Super 8× TOPFlash purchased from Addgene (#12456), and codon-optimized tandem (td) IRFP ordered from Twist Biosciences as overlapping gene fragments with the sequences:PiggyBacPuro backbone fwd: ACCTGCGGGGACGTGGAAGAAAACCCGGGTCCAatgaccgagtacaagcccacggtg,rev: cattccacagggtcgacagtacaagcaaaaag.8× TOPFlash fwd: cttgtactgtcgaccctgtggaatgaagtgcaggtgccagaacatttctc,rev: GTCGGGCCACGCTGCCTTCAGCCATggtggctttaccaacagtaccgg.tdIRFP1 fwd: ATGGCTGAAGGCAGCGTGGC,rev: GGAGGTGCCACTAGAGCTGC. tdIRFP2fwd: TAGCGGCAGCTCTAGTGGCAC,rev: GGTTTTCTTCCACGTCCCCGCAGGTCAACAAACTTCCGCGACCTTCTCCGCTCCCCTCCTCCATTACCCCTATCTGCCAAGCGPCR fragments were amplified using the following primers:E. coli Transformation Kit and Buffer Set (Zymo Research #T3002), cultured on lysogeny broth agar plates to select for antibiotic resistance using standard workflows for molecular cloning and DNA production  and pMD 2.G at a 1:0.88:0.11 mass ratio using standard PEI-based transfection procedures (50). Cells were incubated for 24 h before replacing with fresh media and allowing for lentiviral production for an additional 48 h. Supernatant was harvested, filtered through a 0.22-μm filter and added to plated cells for transduction. Note that all steps for lentiviral production, transduction, and subsequent maintenance of cell lines were carried out in the presence of far-red light or the complete absence of light in an attempt to eliminate the possibility of Cry-2 opto-GSK3 clustering interference with cell growth or virus production.Genomic edits in 293Ts were carried out in cells constitutively expressing Cas9 to maximize editing efficiency.fwd: acgcgccctgtagcgrev: cttaatgcgccgctacagggcgcgtggtacctctagagccatttgtctgc,assembled, cloned, purified, and verified as described in the previous section. The baseline pCab_minimal construct was then subsequently used for production of gRNAs targeting exon1 of the human genomic loci of CTNNB1, CSNK1a1, and GSK3B. Primers creating one to three (depending on protospacer adjacent motif site availability/predicted on/off-target editing scores) unique protospacers targeting the 50-bp window surrounding the first codon of each gene were annealed and cloned into the pCAB_minimal via BbsI digestion and ligation  using standard protocols (CTNNB1_1 fwd: caccgTGAGTAGCCATTGTCCACGC rev: aaacGCGTGGACAATGGCTACTCACTNNB1_2 fwd: caccgTGAAAATCCAGCGTGGACAA rev: aaacTTGTCCACGCTGGATTTTCAcCTNNB1_3 fwd: caccGCGTGGACAATGGCTACTCA rev: aaacTGAGTAGCCATTGTCCACGCCSNK1a1_1 fwd: caccGGCCAAGCCCCGACACCTCT rev: aaacAGAGGTGTCGGGGCTTGGCCCSNK1a1_2 fwd: caccgAGGCTGAATTCATTGTCGGA rev: aaacTCCGACAATGAATTCAGCCTGSK3B fwd: caccCGAAGAGAGTGATCATGTCA rev: aaacTGACATGATCACTCTCTTCGA vector expressing guide RNA (gRNA) and Cas9 obtained from M.Z.W. was subcloned to remove the unnecessary Cas9 ORF via PCR using the following primers:rotocols . The folAXIN1 gRNAs were ordered complete from IDT. Four different protospacer sequences were used (in separate reactions) with the same homology-directed repair (HDR) template to maximize chance of target locus cutting. Cells from each reaction were then pooled 7 d after transfection and subsequently enriched together.AXIN1_1: GGCCGTCCTGCCCGTCTTTGAXIN1_2: GTCTTTGAGGAGAAGATCATAXIN1_3: gTCTTTGAGGAGAAGATCATCAXIN1_4: GGAGAAGATCATCGGCAAAG.Protospacer sequences:tdmRuby3: GGcGGTAGTGGcGGcGGAagcATGGTTAGCAAAGGGGAGGAGCTTATAAAGGAAAATATGAGAATGAAAGTTGTCATGGAAGGTTCAGTGAATGGCCATCAGTTTAAATGTACAGGTGAAGGCGAGGGACGCCCTTATGAAGGAGTCCAAACTATGAGGATCAAAGTCATAGAGGGAGGTCCTCTCCCCTTCGCCTTCGATATCCTCGCCACCTCTTTCATGTATGGTTCAAGAACATTTATCAAGTATCCTGCCGATATACCAGACTTCTTTAAGCAGTCATTTCCAGAAGGTTTCACTTGGGAACGAGTCACTAGGTATGAGGACGGCGGGGTTGTGACAGTAACTCAAGACACCTCTTTGGAAGATGGTGAGTTGGTCTACAACGTGAAGGTACGCGGGGTTAATTTCCCTTCTAACGGGCCTGTTATGCAAAAGAAGACAAAGGGTTGGGAGCCAAATACCGAGATGATGTATCCTGCAGATGGTGGCCTGCGGGGCTATACCGACATCGCTCTGAAGGTAGACGGCGGGGGCCACCTCCATTGTAATTTTGTAACCACTTACAGGTCTAAGAAGACCGTGGGTAACATTAAGATGCCAGGGGTTCATGCTGTCGACCATAGATTGGAGCGGATAGAAGAAAGCGACAACGAGACCTACGTCGTGCAACGCGAAGTCGCAGTAGCCAAGTATTCCAATCTCGGGGGAGGTATGGATGAACTCTATAAAGGCGGATCCGGTGGTGTGTCCAAGGGAGAAGAACTGATCAAAGAGAACATGAGGATGAAGGTCGTGATGGAGGGCAGCGTCAACGGACACCAATTCAAGTGCACCGGAGAGGGAGAAGGCAGACCATACGAGGGCGTGCAGACAATGAGAATTAAGGTGATCGAAGGCGGACCACTGCCTTTTGCTTTCGACATTCTGGCTACAAGCTTCATGTACGGCAGCAGGACCTTCATTAAATACCCCGCTGACATCCCTGATTTTTTCAAACAAAGCTTCCCTGAGGGCTTTACCTGGGAGAGAGTGACAAGATACGAAGACGGAGGCGTCGTCACCGTCACACAGGATACAAGCCTGGAGGACGGAGAACTGGTGTATAACGTCAAAGTCAGAGGAGTGAACTTTCCCAGCAATGGCCCCGTGATGCAGAAAAAGACCAAAGGCTGGGAACCTAACACAGAAATGATGTACCCAGCCGACGGAGGACTGAGAGGATACACAGACATTGCCCTCAAAGTGGATGGAGGAGGACATCTGCACTGCAACTTCGTCACAACCTACAGATCCAAGAAAACAGTCGGAAATATCAAGATGCCTGGCGTGCACGCCGTGGATCACAGGCTGGAAAGGATTGAGGAGTCCGATAATGAAACATATGTGGTCCAGAGGGAGGTGGCCGTCGCTAAATACAGCAACCTGGGCGGCGGCATGGACGAGCTGTACAAGGGGGGATCAGGAGGaGGctctCTNNB1 fwd: ATAAAAAGACATTTTTGGTAAGGAGGAGTTTTCACTGAAGTTCAGCAGTGATGGAGCTGTGGTTGAGGTGTCTGGAGGAGACCATGAGGTCTGCGTTTCA CTAACCTGGTAAAAGAGGATATGGGTTTTTTTTGTGGGTGTAATAGTGACATTTAACAGGTATCCCAGTGACTTAGGAGTATTAATCAAGCTAAATTTAAATCCTAATGACTTTTGATTAACTTTTTTTAGGGTATTTGAAGTATACCATACAACTGTTTTGAAAATCCAGCGTGGACAGGcGGTAGTGGcGGcGGAagcrev: TAGGGAACCACCTAACAGTTACTCACTGAATCAGTGGAAGAATGGTACTGCATCCAGGCTCCAGAAGCAGTCATCCAGACTAGATTCCTGCTGGTGGCTT GTTTGCTATTTCACCAAGCCATTAGGAGGAGTGAGCAGAAAATGGAGCAAAAGGTAGCCTGACAAGTAAGCAGGGAGAGAGGAAAGCAGGGGGATCTCAGCCAGACTGGCTTAATGGCAACGAAGCAGAGCCCCAATTCAGTAACTAAAGATTTAATGACACAAACCTTGAGTAGCCATagagCCtCCTCCTGATCCCCCBlunt-end PCR products were used in conjunction with gRNAs to template genomic edits containing desired knock-ins. Blunt-end, double-stranded HDR templates were created from templates obtained via DNeasy Blood and Tissue genomic prep  of the 293T cell line to be edited (see next section). PCR was conducted using primers targeting amplicons of a 500- to 1,000-bp window centered on the intended cut site. The following primers were used to amplify genomic loci homology regions:CSNK1a1fwd: CCAGCCCGCGACGTC rev: CTTGACCCTTTTAGGGAGACAGCGGSK3B fwd: GATTTGCCCTCTCTTTTCTCTCCTCC rev: CCAAATAAATATCATATTATCTCAATTCAAGGTTAATGAGACCGNote that CTNNB1 homology arms were synthesized (requiring no genomic amplification step) and provided as a generous gift from Integrated DNA Technologies.Generic tdmRuby3 insert fwd: GGcGGTAGTGGcGGcGGAagcATGGTTAGCAAAGGGGAGGAGC, rev: agagCCtCCTCCTGATCCCCCCTTGTACAGCTCGTCCATGCCCSNK1a1 upstream homology arm rev: gctTCCgCCgCCACTACCgCCCCTGAGAGACGAAGATGGAGGCCSNK1a1 downstream homology arm fwd: GGGGGATCAGGAGGaGGctctATGGCGAGTAGCAGCGGCGSK3B upstream homology arm rev: gctTCCgCCgCCACTACCgCCGATCACTCTCTTCGCGAATCACCGSK3B downstream homology arm fwd: GGGGGATCAGGAGGaGGctctATGTCAGGGCGGCCCAXIN1 upstream homology arm fwd: CTTCACCCACATGTGGTCATTGCACAXIN1 upstream homology arm rev: CGGCAAAGTGGAGAAGGTGGACGGcGGTAGTGGcGGcGGAagcAXIN1 downstream homology arm fwd: GGGGGATCAGGAGGaGGctctTGATAGGCTGGTGGGCTGGCCAXIN1 downstream homology arm rev: CACCTGAAGCTGGCAGCAGGThe above amplicons were then used in a second round of PCR to obtain separate upstream and downstream homology arms that flanked desired knock-ins, and overlap extension was used to construct the final desired amplicons bearing tdmRuby3 and 7AA GS linker. The following primers were used:Note that original upstream fwd and downstream rev primers listed above for isolating genomic loci were reused in the present step and thus not repeated here.Bare 293T cells were first cotransfected using polyethyleniminePEI  with theCRISPR chassis cells were then cotransfected with one of the constructed gRNA plasmids and respective HDR templates at a 2:1 HDR template:gRNA plasmid molar ratio and allowed 72 h to reach steady-state expression. Similar to the process described above, cells were subject to two rounds of FACS  to obtain a clonal population. Knock-in validation was accomplished via a combination of fluorescence microscopy, genomic PCR, and sequencing . In the case of all intended knock-ins, spatiotemporal fluorescence expression of cell populations was binary (either fluorescent or not) and uniform , suggesting that selected clones were broadly representative of overall edited populations.The 293Ts were cotransfected as described in the previous section with PiggyBac and compatible XLone-Axin-tdmRuby3 and pPig_CuO-APC-tdmIRFP670::CymR expression cassettes. Seventy-two hours after transfection, cells were selected in 1 μM Blasticidin  and 100 μg/mL Hygromycin B Gold . Blast+/Hygro+ cells were then clonally sorted via FACS as described in the previous section to obtain a uniform population for experiments. For iPSCs, Both Piggyback and Donor plasmids were chemically transfected when cells reached 30% confluency using Lipofectamine Stem Transfection Reagent (manufacturer’s protocol). Following transfection, Blasticidin selection (1 μM) was initiated 5 d later. At the end of Blasticidin selection, 12 clones were manually picked under a dissection microscope and continuously cultured in Blasticidin (1 μM) for an additional week. Upon fluorescence signal confirming successful integration, Blasticidin (1 μM) treatment ceased, and one clone was chosen for the remaining experiments.CHIR 99021 (STEMCELL Technologies #72052) was resuspended in DMSO according to supplied manufacturer recommendations and diluted to 5× concentrated stocks in culture medium immediately prior to use on cells. In all cases, CHIR was used at 10 μM. Doxycycline hyclate (Sigma Aldrich #D9891-1G) was resuspended in phosphate-buffered saline (PBS) and diluted to 5× desired concentration in culture medium prior to use. Stock cumate solution (System Biosciences #QM100A-1) was diluted to 5× in culture medium prior to use.SI Appendix, Fig. S4 I and J was 100 ng/mL.The “Low” dose of Dox referred to in Recombinant Human Wnt-3a (R&D Systems 5036-WN-010) was resuspended in in PBS containing 0.1% bovine serum albumin according to supplied manufacturer recommendations and diluted to 5× concentration in culture medium immediately prior to use. In all cases, Wnt-3a was used at a final concentration of 1 μg/mL.Primary antibodies used for immunofluorescent markers of the centrosome were α-GM130  and α-γ-tubulin . The secondary used for both stains was α-Ms-Alexa-488 . Tissue fixation and staining was carried out using standard protocols using cold methanol . Immunof2. Glass-bottom culture plates (Cellvis #P96-1.5H-N) were pretreated with bovine fibronectin (Sigma #F1141) in the case of 293Ts or Matrigel in the case of H9 and iPSCs, and cells were allowed to adhere to the plate before subsequent treatment or imaging. FRAP was performed via custom Nikon NIS Elements JOBs function and 488-nm FRAP laser .All live and fixed cell imaging experiments were carried out using a Nikon W2 SoRa spinning-disk confocal microscope equipped with incubation chamber maintaining cells at 37 °C and 5% COSpatial patterning of light during timelapse fluorescent imaging sessions was accomplished via purpose-built microscope-mounted LED-coupled digital micromirror devices (DMDs) triggered via Nikon NIS Elements software. Stimulation parameters  were optimized to minimize phototoxicity while maintaining continuous activation of Cry-2. For DMD-based stimulation on the microscope, the final settings for “Light ON” were 25% LED power (λ = 455 nm), 2-s duration pulses every 30 s. For experiments that did not require frequent confocal imaging, cells were stimulated via a benchtop LED array purpose built for light delivery to cells in standard tissue culture plates (“OptoPlate”) adapted from previously established designs . The samAll quantification of raw microscopy images was carried out using the same general workflow: background subtraction > classification > measurement > normalization > statistical comparison. Subcellular segmentation of nuclear fluorescence was performed via custom Matlab scripts using H2B-mTagBFP2 brightness, size, and circularity to mask objects. When experimental conditions did not permit segmentation via H2B-mTagBFP2 nuclear fluorescence (such as with live-cell optogenetic stimulation), cells were selected at random using custom ImageJ macro that generates random regions of interest (ROIs) (available upon request). Unless otherwise noted, mean fluorescent intensity of ROIs were measured and subsequently processed. Raw measurements were compiled, processed, and plotted via custom Matlab scripts, available upon request.Statistical parameters are provided in https://fenicsproject.org/) to solve the modified Cahn–Hilliard partial differential equations using the finite element method. In our simulation, we represent the volume fraction of each DC protein, SI Appendix, Fig. S3B), and Ri is the added reaction term such thatWe used the Python-based FEniCS computing environment . We generate a grid mesh with closed boundary conditions to mimic the closed system within a cell. A layer is generated for each simulated component, and ±5% noise of the initial value is added to induce inhomogeneities. The FEniCS package partial differential solver is called to generate the chemical potential with respect to each component. The final step is to define the output file path and then use the built-in Newton solver to generate the simulation. The simulations are then rendered using Paraview software. A detailed Python notebook of the simulations is available on https://github.com/MZWLab/Lach2022.First, all parameters are defined . This allowed us to test the sensitivity of a single metric to alterations in our model’s parameters. In Movie S6) and also increased the nucleation efficiency.We defined the nucleation efficiency of β-cat processing for a given simulation by comparing the ratio of the integrated P4-β-cat to β-cat between identical simulations with and without a nucleator (Supplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary File


Ctenidae Keyserling, 1877 has a worldwide distribution with 584 species belonging to 49 genera. Amongst these, 141 species are from Asia, including 130 species assigned to Cteninae Keyserling, 1877.The spider family Cteninae are reported from Asia: Amauropelmakrabi sp. n. , Am.phangnga sp. n. , Am.saraburi sp. n. ; Anahitamedog sp. n. ; Bowieninhbinh sp. n.  and B.vinhphuc sp. n.  from the robustus-species group; B.borneo sp. n.  from the chinagirl-species group; B.engkilili sp. n. ; B.sabah sp. n.  from the scarymonsters-species group. The male of An.popa Jäger & Minn, 2015 and the female of B.fascination Jäger, 2022 (robustus-species group) are described for the first time. B.fascination Jäger, 2022 is reported from China for the first time. In addition, the DNA barcodes of all the species in this study were obtained, except for B.vinhphuc sp. n.Nine new species belonging to three genera of  Ctenidae Keyserling, 1877 has a worldwide distribution, but mainly occurs in the tropical and subtropical regions   . Both Amrom Asia .Amauropelma, Anahita and Bowie are described from Asia. This brings the total number of Ctenidae to 150 species in Asia, of which 139 species belong to Cteninae . Photos were stacked with Helicon Focus® (Version 7.6.1) or Zerene Stacker® (Version 1.04) and processed in Adobe Photoshop CC2019®. All measurements are in millimetres (mm) and were obtained with an Olympus SZX16 stereomicroscope with a Zongyuan CCD industrial camera. Total length does not include the chelicerae. Palp and leg measurements are shown as: total length . Leg segments were measured on their dorsal side. The distribution map was generated with ArcGIS 10.2 (ESRI Incorporated Company). References to figures in the cited papers are listed in lowercase (fig. or figs); figures from this paper are noted with a capital letter . The type material is deposited in the Institute of Zoology, Chinese Academy of Sciences (IZCAS) in Beijing, China.Size classes are used according to Terminology and taxonomic descriptions follow B.vinhphuc sp. n. A partial fragment of the mitochondrial cytochrome oxidase subunit I (COI) gene was amplified and sequenced, using the following primers: forward: LCO1490 (5’-CWACAAAYCATARRGATATTGG-3’) and reverse: HCO2198 (5'-TAAACTTCAGGGTGACCAAAAAATCA-3')  and reverse: HCO2198 (5'-TAAACTTCAGGGTGACCAAAAAATCA-3')  , except ATCA-3') . COI p-dATCA-3') . For addS. Li & Yaosp. n.C5E9E60C-A668-5AE7-A9FF-89D05F8A802757EA8144-E44C-4F59-8B3A-0D16719CB5D5Type status:Holotype. Occurrence: recordedBy: Z. Chen; individualCount: 1; sex: female; lifeStage: adult; Taxon: order: Araneae; family: Ctenidae; genus: Amauropelma; Location: country: Thailand; stateProvince: Krabi; verbatimLocality: Ao Luk District, Klang Cave; verbatimElevation: 36 m a.s.l.; verbatimLatitude: 8°20.268'N; verbatimLongitude: 98°44.707'E; Event: year: 2015; month: 10; day: 12; Record Level: institutionCode: IZCAS-Ar 43530MaleUnknown.Female (IZCAS-Ar 43530): PL 3.3, PW 2.4, AW 1.6, OL 3.1, OW 1.6. Eye diameters and interdistances: AME 0.10, ALE 0.13, PME 0.11, PLE 0.11, AME–AME 0.04, AME–ALE 0.11, PME–PME 0.06, PME–PLE 0.24, AME–PME 0.06, ALE–PLE 0.08, clypeus AME 0.12, clypeus ALE 0.17. Palp and leg measurements: palp 3.8 , I 10.3 , II 9.2 , III 9.0 , IV 12.2 . Leg formula 4123. Spination of palp and legs: palp 130, 100, 1111, 1212; femora I p002, d111, r010, II p010, d111, r010, III p111, d111, r012, IV p002, d111, r102; patellae I–IV 001; tibiae I–II v22222, III p11, d111, r11, v222, IV p111, d11, r11, v222; metatarsi I–II v222, III p112, d010, r112, v222, IV p112, r112, v222. Chelicerae with 3 promarginal, 4 + 1 retromarginal teeth, without denticles. Retromargin of chelicerae close to fang base without bristle. Tarsi and metatarsi without scopula. Claw tufts arising separately, but intermingle with each other distally. Palpal claw with 3 secondary teeth, leg claws I–II with 3, III with 2 and IV with 3 secondary teeth. Position of tarsal organ: I 0.76, II 0.72, III 0.68.Copulatory organ Fig. a and b. Colour Fig. c and d. Ctenidae . The new species can be distinguished from all known congeners by the median plate roughly heart-shaped and with a mating plug :OP561682).TGTTTGGAGCTTGAGCTGCTATAGCAGGAACTGGAATAAGAGTGTTGATTCGAATAGAGTTAGGTCATCCTGGTAGATTGTTAGGAGATGATCATTTATATAATGTTATTGTAACTGCTCATGCTTTTGTAATGATTTTTTTTATAGTAATACCAATTTTGATTGGTGGATTTGGAAATTGATTAGTTCCGTTGAGATTGGAGCACCTGATATATCATTTCCTCGAATAAATAATTTGTCGTTTTGATTACTACCTCCTTCTTTATTTTTATTAATAATATCATCAATAGTAGAAATAGGTGTTGGAGCGGGATGAACTGTTTATCCTCCTTTAGCATCTAGTATTGGGCATATAGGAAGATCTATAGATTTTGCTATTTTTTCTCTTCATTTGGCTGGAGCTTCTTCTATTATAGGAGCAGTAAATTTTATTTCTACTATTATTAATATACGGTTGTATGGAATGAGTATAGAAAAGGTTCCTTTGTTTGTGTGGTCTGTTTTTATTACTGCTATTTTGTTATTATTGTCGTTACCTGTGTTAGCAGGTGCTATTACTATATTATTGACTGATCGAAATTTTAATACTTCTTTTTTTGACCCTGCGGGAGGGGGAGATCCTATTTTGTTTCAACATTTATTTTGATTTTTTG : PL 3.3, PW 2.8, AW 1.2, OL 3.0, OW 1.9. Eye diameters and interdistances: AME 0.09, ALE 0.12, PME 0.10, PLE 0.10, AME–AME 0.04, AME–ALE 0.13, PME–PME 0.08, PME–PLE 0.21, AME–PME 0.05, ALE–PLE 0.08, clypeus AME 0.17, clypeus ALE 0.22. Palp and leg measurements: palp 3.8 , I 13.5 , II 11.6 , III 10.9 , IV 14.8 . Leg formula 4123. Spination of palp and legs: palp 131, 100, 1101; femora I p021, d211, r112, II–III p012, d111, r012, IV p102, d111, r012; patellae I–IV 001; tibiae I p010, v22222, II p100, r100, v22222, III p11, d111, r11, v222, IV p11, d11, r11, v222; metatarsi I v222, II p112, r010, v222, III p112, d010, r112, v222, IV p112, r112, v2222. Chelicerae with 3 promarginal, 4 retromarginal teeth, without denticles. Retromargin of chelicerae close to fang base without bristle. Tarsi and metatarsi without scopula. Claw tufts arising separately, but intermingle with each other distally. Leg claws I with 7 and II with 6 secondary teeth. Position of tarsal organ: I 1.37, II 0.92, III 0.85.Palp Fig. a–c. Pate9Colour Fig. a and b. FemaleUnknown.Variation: Paratype male (IZCAS-Ar 43532): PL 3.4, OL 2.4.Ctenidae . The new species can be distinguished from all known congeners by the embolus tip with an extension :OP718556).GGTGGGTTCGGAAATTGATTGGTTCCTTTGATGTTAGGAGCTCCTGATATATCATTTCCTCGTATAAATAATTTGTCTTTTTGGTTACTTCCTCCTTCTTTATTTTTGTTATTAATATCTTCTATGGTGGAAATAGGAGTGGGAGCAGGATGAACTGTCTATCCTCCTTTAGCTTCTAGAATAGGGCATGTGGGAAGATCAATAGATTTTGCGATTTTTTCTCTTCATTTAGCTGGAGTTTCTTCTATTATGGGAGCGGTTAATTTTATTTCTACTATTATTAATATGCGATTATATGGAATAACTATAGAAAAGGTTCCTTTATTCGTTTGATCAGTTTTTATTACTGCAGTTTTGTTGTTGTTATCATTACCTGTGTTAGCAGGTGCTATTACTATATTATTGACAGATCGAAATTTTAATACTTCTTTTTTTGATCCTGCAGGGGGTGGAGATCCAATTTTATTTCAACATTTATTCTGATTTTTTGGTCACCCTGGAAAGTTTAA : PL 4.5, PW 3.7, AW 1.6, OL 3.2, OW 2.1. Eye diameters and interdistances: AME 0.12, ALE 0.14, PME 0.14, PLE 0.14, AME–AME 0.07, AME–ALE 0.16, PME–PME 0.08, PME–PLE 0.26, AME–PME 0.07, ALE–PLE 0.13, clypeus AME 0.16, clypeus ALE 0.28. Palp and leg measurements: palp 5.2 , I - , II 15.2 , III 14.3 , IV 19.4 . Leg formula 4123. Spination of palp and legs: palp 131, 100, 210; femora I p112, d111, r111, II p211, d111, r211, III p112, d111, r112, IV p112, d111, r002; patellae I 001, II–IV 101; tibiae I p010, r110, v22222, II p100, r100, v22222, III–IV p11, d111, r11, v222; metatarsi I v222, II p110, r110, v222, III p112, d010, r112, v222, IV p112, d010, r112, v2222. Chelicerae with 3 promarginal, 4 retromarginal teeth, without denticles. Retromargin of chelicerae close to fang base without bristle. Claw tufts arising separately, but intermingle with each other distally. Leg claws II with 1 and III–IV with 2 secondary teeth. Position of tarsal organ: IV 1.58.Palp Fig. a–c. Pate9Colour Fig. c and d. Female (IZCAS-Ar 43534): PL 5.6, PW 4.4, AW 2.8, OL 4.9, OW 3.1. Eye diameters and interdistances: AME 0.14, ALE 0.20, PME 0.15, PLE 0.16, AME–AME 0.12, AME–ALE 0.32, PME–PME 0.16, PME–PLE 0.53, AME–PME 0.08, ALE–PLE 0.20, clypeus AME 0.13, clypeus ALE 0.21. Palp and leg measurements: palp 6.0 , I 19.3 , II 18.3 , III 17.0 , IV 23.0 . Leg formula 4123. Spination of palp and legs: palp 131, 100, 1111, 2112; femora I p021, d111, r111, II–III p112, d111, r112, IV p112, d111, r002; patellae I–II 000, III–IV 101; tibiae I –II v22222, III–IV p11, d111, r11, v222; metatarsi I–II v222, III–IV p112, d010, r112, v222. Chelicerae with 3 promarginal, 4 retromarginal teeth, without denticles. Retromargin of chelicerae close to fang base without bristle. Sparse scopula restricted almost entirely to tarsi, only metatarsi I–II with sparse scopula hairs. Claw tufts arising separately, but intermingle with each other distally. Palpal claw with 3 secondary teeth, leg claws I with 3, II–III with 2 secondary teeth.Copulatory organ Fig. a and b. Colour Fig. e and f. Variation: Second paratype female (IZCAS-Ar 43535): PL 4.6, OL 4.7.Ctenidae . The new species can be distinguished from all known congeners by the embolus slender .Male (IZCAS-Ar 43533):TGTTTGGAGCTAGATCTGCTATAGCGGGAACGGCAATAAGAGTTTTAATTCGTATGGAATTAGGAAATTCTGGAAGATTATTAGGGGATGATCATTTATATAATGTAATTGTGACAGCTCATGCTTTTATTATGATTTTTTTTATAGTAATACCGATTTTGATTGGTGGTTTTGGAAATTGATTAGTGCCTTTAATGTTAGGAGCTCCTGATATATCTTTTCCTCGGATGAATAATTTGTCTTTTTGATTACTTCCACCTTCTTTGTTTTTATTATTCATATCTTCTATGGTGGAAATGGGTGTAGGAGCTGGATGAACTGTTTATCCACCTTTGGCTTCTAGAATTGGTCATGCTGGAAGATCTATGGATTTTGCTATTTTTTCTTTACATTTAGCTGGGGCTTCTTCAATTATAGGAGCGGTGAATTTTATTTCTACTATTATTAATATACGATTATCTGGAATAAGAATGGAGAAGGTTCCATTATTTGTTTGATCTGTTCTTATTACTGCAATTTTATTATTATTATCTTTGCCGGTATTAGCTGGTGCTATTACTATATTGTTGACTGATCGAAATTTTAATACTTCTTTTTTTGATCCGGCTGGGGGAGGGGATCCTATTTTATTTCAACATTTATTTTGATTTTTTG (GenBank accession number OP572099).Female (IZCAS-Ar 43534):TGTTTGGAGCTTGATCTGCTATAGCGGGAACGGCAATAAGAGTTTTAATTCGTATGGAATTAGGAAATTCTGGAAGATTATTAGGGGATGATCATTTATATAATGTAATTGTGACAGCTCATGCTTTTATTATGATTTTTTTTATAGTAATACCGATTTTGATTGGTGGTTTTGGAAATTGATTAGTGCCTTTAATGTTAGGAGCTCCTGATATATCTTTTCCTCGGATGAATAATTTGTCTTTTTGATTACTTCCACCTTCTTTGTTTTTATTATTCATATCTTCTATGGTGGAAATGGGTGTAGGAGCTGGATGAACTGTTTATCCACCTTTGGCTTCTAGAATTGGTCATGCTGGAAGATCTATGGATTTTGCTATTTTTTCTTTACATTTAGCTGGGGCTTCTTCAATTATAGGAGCGGTGAATTTTATTTCTACTATTATTAATATACGATTATCTGGAATAAGAATGGAGAAGGTTCCATTATTTGTTTGATCTGTTCTTATTACTGCAATTTTATTATTATTATCTTTGCCGGTATTAGCTGGTGCTATTACTATATTGTTGACTGATCGAAATTTTAATACTTCTTTTTTTGATCCGGCTGGGGGAGGGGATCCTATTTTATTTCAACATTTATTTTGATTTTTTG : PL 2.7, PW 2.3, AW 0.9, OL 2.4, OW 1.4. Eye diameters and interdistances: AME 0.13, ALE 0.10, PME 0.22, PLE 0.19, AME–AME 0.10, AME–ALE 0.22, PME–PME 0.16, PME–PLE 0.23, AME–PME 0.11, ALE–PLE 0.14, clypeus AME 0.10, clypeus ALE 0.37. Palp and leg measurements: palp 4.1 , I missing, II 13.0 , III 10.9 , IV 16.0 . Leg formula 4123. Spination of palp and legs: palp 023, 000, 0211; femora II p112, d111, r012, III p112, d111, r112, IV p112, d111, r012; patellae II–IV 101; tibiae II p110, d101, r100, v22222, III p11, d111, r11, v222, IV p11, d111, r11, v22; metatarsi II p111, r111, v222, III p112, d010, r112, v222, IV p112, d010, r112, v2222. Chelicerae with 3 promarginal, 4 + 1 retromarginal teeth and with elongated patch of 6 tiny denticles along entire cheliceral furrow. Leg claws II with 9, III with 5 and IV with 7 secondary teeth. Position of tarsal organ: II 1.06, III 0.85, IV 1.14.Palp Fig. a–c. Palp14Colour Fig. c and d. Female (IZCAS-Ar 43537): PL 2.9, PW 2.4, AW 1.2, OL 3.5, OW 2.0. Eye diameters and interdistances: AME 0.13, ALE 0.11, PME 0.19, PLE 0.18, AME–AME 0.13, AME–ALE 0.26, PME–PME 0.21, PME–PLE 0.24, AME–PME 0.15, ALE–PLE 0.17, clypeus AME 0.10, clypeus ALE 0.38. Palp and leg measurements: palp 3.2 , I 10.4 , II 9.3 , III 8.3 , IV 12.4 . Leg formula 4123. Spination of palp and legs: palp 020, 010, 010, 2012; femora I p011, d111, r021, II p011, d111, r011, III p012, d111, r112, IV p002, d111, r012; patellae I–II 000, III–IV 101; tibiae I v22212, II v22222, III–IV p11, d111, r11, v222; metatarsi I–II v222, III p112, d010, r112, v222, IV p112, r112, v2222. Chelicerae with 3 promarginal, 4 retromarginal teeth and with elongated patch of 5 tiny denticles along entire cheliceral furrow. Palpal claw with 5 secondary teeth, leg claws I–II with 5, III with 4 and IV with 7 secondary teeth. Position of tarsal organ: I 0.79, II 0.72, III 0.70, IV 1.00.Copulatory organ Fig. a and b. Colour Fig. e and f. Ctenidae . The species resembles A.maolan Zhu, Chen & Song, 1999 (see 14A.maolan), by the median plate with a large n-shaped sclerite , by the lateral teeth pointing postero-medially  and by the spermathecae nearly cylindrical .Small 1999 see a–c and f1999 see b, but caThe specific name refers to the type locality and is a noun in apposition.China :.OP572101).TGTTTGGAGCTTGAGCTGCTATAGCTGGAACAGCAATAAGAGTTTTAATTCGAATGGAATTAGGACATTCTGGTAGATTGTTAGGAGATGATCATTTATATAATGTAATTGTAACGGCTCATGCTTTTGTTATAATTTTTTTTATAGTAATACCTATTTTGATTGGGGGCTTTGGTAATTGGTTGGTTCCTTTAATGTTAGGGGCTCCGGATATATCTTTTCCTCGAATAAATAATTTATCCTTTTGATTATTACCGCCTTCTTTATTTTTGTTGTTTATATCTTCTATAGTTGAGATAGGGGTTGGAGCAGGTTGAACGGTTTATCCTCCTTTAGCTTCTAGAATTGGGCATATGGGAAGTTCAATGGATTTTGCTATTTTTTCTTTACATTTAGCAGGTGCTTCTTCTATTATAGGTGCTGTGAATTTTATTTCTACTATTATTAATATACGATTAATAGGAATAACAATGGAGAAGATCCCTTTATTTGTATGATCGGTTTTTATTACTGCAATTTTATTATTATTATCTTTACCTGTTTTAGCAGGAGCTATTACTATATTATTGACTGATCGAAATTTTAATACTTCTTTTTTTGACCCTGCTGGAGGTGGAGATCCTATTTTATTTCAACATTTATTTTGATTTTTTG :OP572102).TGTTTGGAGCTTGAGCTGCTATAGCTGGAACAGCAATAAGAGTTTTAATTCGAATGGAATTAGGACATTCTGGTAGATTGTTAGGAGATGATCATTTATATAATGTAATTGTAACGGCTCATGCTTTTGTTATAATTTTTTTTATAGTAATACCTATTTTGATTGGGGGCTTTGGTAATTGGTTGGTTCCTTTAATGTTAGGGGCTCCGGATATATCTTTTCCTCGAATAAATAATTTATCCTTTTGATTATTACCGCCTTCTTTATTTTTGTTGTTTATATCTTCTATAGTTGAGATAGGGGTTGGAGCAGGTTGAACGGTTTATCCTCCTTTAGCTTCTAGAATTGGGCATATGGGAAGTTCAATGGATTTTGCTATTTTTTCTTTACATTTAGCAGGTGCTTCTTCTATTATAGGTGCTGTGAATTTTATTTCTACTATTATTAATATACGATTAATAGGAATAACAATGGAGAAGATCCCTTTATTTGTATGATCGGTTTTTATTACTGCAATTTTATTATTATTATCTTTACCTGTTTTAGCAGGAGCTATTACTATATTATTGACTGATCGAAATTTTAATACTTCTTTTTTTGACCCTGCTGGAGGTGGAGATCCTATTTTATTTCAACATTTATTTTGATTTTTTG : PL 3.3, PW 2.7, AW 0.9, OL 3.3, OW 1.8. Eye diameters and interdistances: AME 0.14, ALE 0.12, PME 0.22, PLE 0.17, AME–AME 0.08, AME–ALE 0.23, PME–PME 0.16, PME–PLE 0.21, AME–PME 0.18, ALE–PLE 0.13, clypeus AME 0.10, clypeus ALE 0.55. Palp and leg measurements: palp 3.9 , I 15.4 , II 12.7 , III 11.0 , IV 17.3 . Leg formula 4123. Spination of palp and legs: palp 151, 000, 122; femora I p021, d111, r112, II p112, d111, r112, III p111, d111, r111, IV p012, d111, r112; patellae I–IV 101; tibiae I p010, d101, r110, v22222, II p10, d101, r110, v22222, III–IV p11, d111, r11, v222; metatarsi I p111, d001, r111, v222, II p111, d111, r111, v222, III p111, d012, r111, v222, IV p112, d011, r112, v222. Chelicerae with 3 promarginal, 4 + 2 retromarginal teeth and with elongated patch of 3 tiny denticles along entire cheliceral furrow. Retromargin of chelicerae close to fang base with 2 bristles. Sparse scopula restricted almost entirely to tarsi. Leg claws I with 6, II–III with 5 and IV with 4secondary teeth. Position of tarsal organ: I 1.89, II 1.03, III 0.75, IV 1.56.Palp Fig. a–c. Palp14Colour Fig. c and d. Female (IZCAS-Ar 43539): See Fig. Ctenidae . The species can be distinguished from all known congeners by the embolus arising at 12 o’clock position, long and laminar, running around tegulum, its tip situated distally :OP572105).TATTTGGGGCTTGAGCTGCTATAGCGGGTACTGCAATAAGAGTTTTGATTCGAATGGAATTAGGACATCCTGGAAGATTATTAGGTGATGATCATTTATATAATGTTATTGTAACAGCTCATGCTTTTGTTATGATTTTTTTTATAGTTATACCTATTTTAATTGGTGGTTTTGGAAATTGGTTAGTTCCTTTAATATTAGGAGCTCCGGATATATCATTTCCTCGAATAAATAATTTATCTTTTTGGTTATTACCTCCTTCTTTGTTTTTATTGTTTATATCTTCTATAGTTGAAATAGGTGTAGGAGCAGGGTGAACAGTTTATCCTCCTTTAGCTTCTAGAATTGGGCATGCAGGGAGATCTATGGATTTTGCTATTTTTTCTTTACATTTAGCGGGTGCTTCTTCTATTATAGGGGCTGTAAATTTTATTTCTACTATTATTAATATACGATTAATAGGAATGACTATAGAGAAGGTTCCTTTGTTTGTTTGATCTGTTTTTATTACTGCAATTTTATTATTGTTATCTTTACCAGTGTTAGCTGGTGCTATTACAATATTATTAACTGATCGTAATTTTAATACTTCTTTTTTTGATCCTGCTGGAGGAGGAGATCCAGTTTTATTTCAGCATTTGTTTTGATTTTTTG :OP572104).TTTTTGGAGCTTGAGCCGCTATAGCGGGTACTGCAATAAGAGTTTTAATTCGAATAGAATTAGGGCATCCTGGGAGATTATTAGGTGATGATCATTTATATAATGTTATTGTAACAGCTCATGCTTTTGTTATAATTTTTTTTATAGTTATACCTATTTTAATTGGTGGTTTTGGAAATTGGTTAGTTCCTTTAATGTTAGGAGCTCCGGATATATCATTTCCTCGAATAAATAATTTATCTTTTTGATTATTACCTCCTTCTTTGTTTTTATTGTTTATATCTTCCATGGTTGAAATAGGTGTGGGAGCAGGATGGACAGTTTATCCTCCTTTAGCTTCTAGAATTGGGCATGCGGGAAGATCTATGGATTTTGCTATTTTTTCTTTACATTTAGCGGGTGCTTCTTCTATTATAGGAGCTGTAAATTTTATTTCGACTATTATTAATATACGATTAATAGGAATGACTATAGAGAAGGTTCCCTTATTTGTTTGATCTGTTTTTATTACTGCAATTTTATTGTTATTATCTTTACCAGTATTAGCTGGTGCTATTACGATGTTGTTAACTGATCGTAATTTTAATACTTCTTTTTTTGACCCTGCTGGGGGAGGGGATCCGGTTTTATTTCAACATTTATTTTGATTTTTTG : See Fig. 28Female (IZCAS-Ar 43541): PL 9.9, PW 7.8, AW 4.1, OL 11.0, OW 7.3. Eye diameters and interdistances: AME 0.29, ALE 0.27, PME 0.32, PLE 0.39, AME–AME 0.31, AME–ALE 0.71, PME–PME 0.43, PME–PLE 1.32, AME–PME 0.32, ALE–PLE 0.38, clypeus AME 0.42, clypeus ALE 0.95. Palp and leg measurements: palp 10.0 , I 23.0 , II 21.4 , III 18.6 , IV 26.1 . Leg formula 4123. Spination of palp and legs: palp 131, 100, 131, 3020; femora I p021, d111, r111, II p112, d111, r111, III–IV p111, d111, r112; patellae I–II 000, III–IV 101; tibiae I–II v22222, III–IV p11, d111, r11, v222; metatarsi I–II v222, III p111, d012, r111, v222, IV p121, d012, r111, v2122. Chelicerae with 3 promarginal, 4 + 1 retromarginal teeth and with elongated patch of 27 tiny denticles along entire cheliceral furrow. Retromargin of chelicerae close to fang base with 11 bristles. Ventral tarsi and metatarsi I–II with sparse scopula. Palpal claw with 5 secondary teeth, leg claws I–II with 1, III with 2 and IV with 3 secondary teeth. Position of tarsal organ: I 1.41, II 1.34, III 1.25, IV 1.44.Copulatory organ Fig. a. EpigynColour Fig. e and f. Ctenidae . The species is assigned to the robustus-species group with the characteristics of stout tegular apophysis, simple stout embolus with broad base, presence of retro-proximal cymbial outgrowth, RTA arising medially to distally from palpal tibia, female possesses a transversally median plate with lateral teeth situated mainly laterally and not reaching the epigastric furrow. It resembles B.candidate Jäger, 2022 (see B.candidate), by the spermathecae separated by less than their diameter and smaller chamber with distinct external rim  and by the fertilisation ducts pointing laterally . For the diagnosis of male, see Large-sized 2022 see a, but caChina :OP572108).TATTTGGATCTTGGGCTGCTATAGCTGGGACAGCTATAAGAGTATTAATTCGTATAGAGCTAGGTCATTCTGGTAGATTATTTGGTGATGATCATTTATATAATGTAATTGTTACAGCTCATGCTTTTGTAATAATTTTTTTTATGGTTATGCCTATTTTAATTGGTGGTTTTGGAAACTGATTAGTTCCTTTGATATTAGGGGCTCCTGATATATCTTTTCCTCGTATAAATAATTTATCTTTTTGATTACTCCCTCCTTCATTATTTTTGTTATTTATATCTTCTATGGTTGAGATAGGGGTGGGAGCTGGTTGGACAGTGTATCCTCCTTTAGCTTCTAGTATTGGCCATATAGGAAGATCAATAGATTTTGCTATTTTTTCTTTACATTTAGCGGGAGCTTCTTCTATTATAGGGGCTGTTAATTTTATTTCTACAATTATTAATATACGTTTGTATGGAGTAAGAATAGAAAAGGTGCCTTTATTTGTATGATCTGTTCTAATTACTGCAGTATTATTGCTTTTATCTTTACCTGTATTAGCAGGTGCTATTACTATATTATTAACTGATCGTAATTTTAATACTTCTTTTTTTGACCCGGCTGGAGGAGGGGATCCAGTTTTATTTCAACATTTATTTTGATTTTTTG :OP572107).TATTTGGATCTTGGGCTGCTATAGCTGGGACAGCTATAAGAGTATTAATTCGTATAGAGCTAGGTCATTCTGGTAGATTATTTGGTGATGATCATTTATATAATGTAATTGTTACAGCTCATGCTTTTGTAATAATTTTTTTTATGGTTATGCCTATTTTAATTGGTGGTTTTGGAAACTGATTAGTTCCTTTGATATTAGGGGCTCCTGATATATCTTTTCCTCGTATAAATAATTTATCTTTTTGATTACTCCCTCCTTCATTATTTTTGTTATTTATATCTTCTATGGTTGAGATAGGGGTGGGAGCTGGTTGGACAGTGTATCCTCCTTTAGCTTCTAGTATTGGCCATATAGGAAGATCAATAGATTTTGCTATTTTTTCTTTACATTTAGCGGGAGCTTCTTCTATTATAGGGGCTGTTAATTTTATTTCTACAATTATTAATATACGTTTGTATGGAGTAAGAATAGAAAAGGTGCCTTTATTTGTATGATCTGTTCTAATTACTGCAGTATTATTGCTTTTATCTTTACCTGTATTAGCAGGTGCTATTACTATATTATTAACTGATCGTAATTTTAATACTTCTTTTTTTGACCCGGCTGGAGGAGGGGATCCAGTTTTATTTCAACATTTATTTTGATTTTTTG : PL 7.6, PW 5.9, AW 3.0, OL 5.8, OW 3.9. Eye diameters and interdistances: AME 0.26, ALE 0.19, PME 0.38, PLE 0.30, AME–AME 0.19, AME–ALE 0.29, PME–PME 0.22, PME–PLE 0.43, AME–PME 0.18, ALE–PLE 0.23, clypeus AME 0.11, clypeus ALE 0.55. Palp and leg measurements: palp 7.8 , I 21.3 , II 19.6 , III 15.9 , IV 23.1 . Leg formula 4123. Spination of palp and legs: palp 151, 100, 101; femora I p021, d111, r112, II p112, d111, r112, III p212, d111, r112, IV p112, d111, r012; patellae I–IV 101; tibiae I p110, d111, r210, v22222, II p110, d111, r110, v22222, III p11, d200, r11, v222, IV p11, d111, r11, v222; metatarsi I–III p112, d010, r112, v222, IV p112, d010, r112, v2222. Chelicerae with 3 promarginal, 4 retromarginal teeth and with elongated patch of 19 tiny denticles along entire cheliceral furrow. Retromargin of chelicerae close to fang base with 6 bristles. Ventral tarsi and metatarsi I–II with sparse scopula. Right leg claws I–III with 2 and IV with 3 secondary teeth. Position of tarsal organ: I 1.27, II 1.28, III 1.06, IV 1.36.Palp Fig. a–c. RTA 28Colour Fig. a and b. FemaleUnknown.Variation: Paratype male (IZCAS-Ar 43543): PL 7.4, OL 5.7.Ctenidae . The new species is assigned to the robustus-species group with the characteristics of stout tegular apophysis, simple stout embolus with broad base, presence of retro-proximal cymbial outgrowth and RTA arising subdistally from palpal tibia. It resembles B.dodo Jäger, 2022 (see B.dodo) and by the RTA distally bifurcated .Medium-sized 2022 see b, but caThe specific name refers to the type locality and is a noun in apposition.Vietnam :OP572110).TACTTGGATCTTGGGCTGCTATGGCAGGGACAGCTATAAGAGTATTAATTCGGATGGAATTAGGCCATTCTGGGAGATTGTTAGGTGATGATCATTTATACAATGTAATTGTTACTGCACATGCTTTTGTAATGATTTTTTTTATAGTAATGCCTATTTTAATTGGGGGTTTTGGAAATTGGTTAGTACCTTTGATATTAGGGGCTCCTGATATATCTTTTCCTCGAATAAATAATTTGTCTTTTTGGTTACTTCCTCCTTCGTTATTTTTATTATTTATATCTTCAATAGTTGAGATAGGAGTTGGAGCTGGATGAACGGTATATCCTCCTTTAGCTTCTAGTATTGGTCATATAGGGAGATCTATAGATTTTGCTATTTTTTCTTTACATTTAGCGGGGGCTTCTTCTATTATAGGAGCGGTAAATTTTATTTCTACGATTATTAATATGCGTTTGTATGGGATGACTATAGAGAAAGTACCTTTATTTGTGTGATCTGTTTTAATTACTGCGGTATTGTTATTATTGTCTTTACCTGTTTTAGCAGGTGCTATTACTATATTGTTAACTGATCGAAATTTTAATACTTCTTTTTTTGATCCGGCTGGGGGTGGTGATCCTGTTTTGTTTCAACATTTATTTTGATTTTTTG : PL 6.4, PW 5.0, AW 2.4, OL 3.1, OW 3.5. Eye diameters and interdistances: AME 0.23, ALE 0.24, PME 0.28, PLE 0.26, AME–AME 0.22, AME–ALE 0.42, PME–PME 0.25, PME–PLE 0.41, AME–PME 0.20, ALE–PLE 0.21, clypeus AME 0.21, clypeus ALE 0.53. Palp and leg measurements: palp 7.4 , I 17.7 , II 15.9 , III 13.4 , IV 19.5 . Leg formula 4123. Spination of palp and legs: palp 141, 100, 1010; femora I p021, d111, r112, II p112, d111, r1111, III p1111, d111, r1111, IV p002, d111, r111; patellae I–IV 101; tibiae I p110, d111, r21, v22222, II p20, d111, r110, v22222, III–IV p11, d111, r11, v222; metatarsi I p11, d002, r111, v222, II p111, r111, v222, III p111, d012, r211, v222, IV p122, d012, r111, v2122. Chelicerae with 3 promarginal, 4 retromarginal teeth and with elongated patch of 12 tiny denticles along entire cheliceral furrow. Retromargin of chelicerae close to fang base with 4 bristles. Sparse scopula restricted almost entirely to tarsi, only metatarsi I–II with sparse scopula hairs. Leg claws I–III with 3 and IV with 4 secondary teeth. Position of tarsal organ: I with 1.17, II 1.07, III 0.90, IV 1.09.Palp Fig. a–c. RTA 28Colour Fig. c and d. Female (IZCAS-Ar 43728): PL 6.7, PW 5.2, AW 3.1, OL 8.5, OW 7.0. Eye diameters and interdistances: AME 0.25, ALE 0.21, PME 0.28, PLE 0.26, AME–AME 0.22, AME–ALE 0.48, PME–PME 0.29, PME–PLE 0.57, AME–PME 0.23, ALE–PLE 0.23, clypeus AME 0.19, clypeus ALE 0.58. Palp and leg measurements: palp 6.9 , I 16.3 , II 15.0 , III 13.1 , IV 18.8 . Leg formula 4123. Spination of palp and legs: palp 131, 000, 1111, 2101; femora I p021, d111, r021, II p112, d111, r021, III p112, d111, r112, IV p111, d111, r001; patellae I–II 000, III–IV 101, tibiae I–II v22222, III–IV p11, d111, r11, v222; metatarsi I–II v222, III p112, d010, r112, v222, IV p112, r112, v2222. Chelicerae with 3 promarginal, 4 retromarginal teeth and with elongated patch of 10 tiny denticles along entire cheliceral furrow. Retromargin of chelicerae close to fang base with 5 bristles. Sparse scopula restricted almost entirely to tarsi, only metatarsi I–II with sparse scopula hairs. Palpal claw with 6 secondary teeth, leg claws I with 1, II–III with 2 and IV with 3 secondary teeth. Position of tarsal organ: I 1.03, II 0.97, III 0.95, IV 1.42.Copulatory organ Fig. b. EpigynColour Fig. e and f. Variation: Paratype male (IZCAS-Ar 43545): PL 7.4, OL 6.5. Second paratype female (IZCAS-Ar 43729): PL 7.6, OL 7.8.Ctenidae . The new species is assigned to the robustus-species group with the characteristics of stout tegular apophysis, simple stout embolus with broad base and short apical part, presence of retro-proximal cymbial outgrowth, RTA arising subdistally from palpal tibia, female possesses a transversally oval median plate with lateral teeth situated laterally and not reaching the epigastric furrow. It resembles B.yassassin Jäger, 2022 (see 28B.yassassin), by the median plate with distinct humped areas best seen in posterior view , by the spermathecae separated by less than their diameter, larger chamber round and apically swollen  and by the fertilisation ducts pointing medially .Small to medium-sized 2022 see a–c and l2022 see b, but caThe specific name refers to the type locality and is a noun in apposition.Vietnam : PL 3.8, PW 2.9, AW 1.2, OL 3.2, OW 2.2. Eye diameters and interdistances: AME 0.17, ALE 0.15, PME 0.22, PLE 0.21, AME–AME 0.14, AME–ALE 0.11, PME–PME 0.24, PME–PLE 0.27, AME–PME 0.11, ALE–PLE 0.13, clypeus AME 0.09, clypeus ALE 0.37. Palp and leg measurements: palp 5.1 , I 12.1 , II 10.9 , III 9.6 , IV 15.1 . Leg formula 4123. Spination of palp and legs: palp 151, 100, 1110; femora I p012, d111, r012, II–III p112, d111, r112, IV p112, d111, r002; patellae I–II 100, III–IV 101; tibiae I p010, r010, v212222, II p100, d001, r110, v22222, III–IV p11, d111, r11, v222; metatarsi I p111, r011, v222, II p111, d002, r111, v222, III p111, d012, r111, v222, IV p111, d012, r111, v2212. Chelicerae with 3 promarginal, 4 retromarginal teeth and with elongated patch of 8 tiny denticles along entire cheliceral furrow. Retromargin of chelicerae close to fang base with 4 bristles. Sparse scopula on all tarsi. Leg claws I–II with 5 and III–IV with 4 secondary teeth. Position of tarsal organ: I 1.07, II 0.89, III 0.72, IV 1.06.Palp Fig. a–c. RTA 28Colour Fig. a and b. FemaleUnknown.Ctenidae . The new species is assigned to the chinagirl-species group with the characteristics of compact embolus, thick-walled, with rounded edges and with an entire margin, the tegular apophysis is longitudinally elongated, not covering the embolus. It resembles B.abdulmajid Jäger, 2022 (see B.abdulmajid), by the tibia having no distinct broad longitudinal ridge ventrally  and by the conductor nearly quadrilateral .Small 2022 see d, but caThe specific name refers to the type locality and is a noun in apposition.Malaysia :OP572103).GGTTTGGTGCTTGGGCTTCTATAGCAGGTACGTCTATAAGAGTTTTGATTCGAATGGAATTAGGACATTCTGGAAGATTATTAGGGGATGATCATTTATATAATGTTGTTGTTACTGCTCATGCTTTTGTTATAATTTTTTTTATAGTGATACCTATTTTAATTGGTGGTTTTGGAAATTGGTTGGTTCCTTTAATATTAGGAGCTCCTGATATATCTTTTCCTCGTATAAATAATTTGTCGTTTTGATTACTTCCTCCTTCTTTATTTTTATTGTTTATATCTTCTATGACTGAGATAGGGGTGGGAGCTGGTTGGACGGTTTATCCACCTTTGGCTTCTGGAATTGGTCATGCAGGAAGATCGATAGATTTTGCTATCTTTTCTCTCCATTTAGCAGGTGCTTCTTCTATTATAGGAGCTATTAATTTTATTTCTACGATTATTAATATACGATTATTAGGAATGAGAATGGAAAAGGTTCCTTTGTTTGTATGGTCTGTTTTTATTACTGCAGTTTTATTATTATTATCTTTACCTGTTTTAGCGGGTGCTATTACTATATTATTAACGGATCGTAATTTTAATACTTCTTTTTTCGATCCTGCTGGAGGAGGGGATCCAATTTTATTTCAACATTTATTTTGATTTTTTG : PL 4.5, PW 3.6, AW 2.3, OL 3.9, OW 2.3. Eye diameters and interdistances: AME 0.21, ALE 0.11, PME 0.26, PLE 0.23, AME–AME 0.15, AME–ALE 0.31, PME–PME 0.27, PME–PLE 0.35, AME–PME 0.13, ALE–PLE 0.17, clypeus AME 0.14, clypeus ALE 0.41. Palp and leg measurements: palp 4.7 , I missing, II 10.8 , III 10.2 , IV 14.4 . Leg formula 4123. Spination of palp and legs: palp 131, 001, 112, 2012; femora II p110, d111, r112, III p012, d111, r112, IV p002, d111, r112; patellae II 000, III–IV 101; tibiae II v22222, III–IV p11, d111, r11, v222; metatarsi II v222, III p112, d010, r112, v222, IV p112, d010, r112, v2222. Chelicerae with 3 promarginal, 4 retromarginal teeth and with elongated patch of 5 tiny denticles along entire cheliceral furrow. Retromargin of chelicerae close to fang base with 5 bristles. Sparse scopula restricted almost entirely to tarsi. Palpal claw with 7 secondary teeth, leg claws II–IV with 3 secondary teeth. Position of tarsal organ: II 0.75, IV 0.85.Copulatory organ Fig. . EpigynaColour Fig. c and d. Ctenidae . The new species resembles B.withinyou Jäger, 2022 (see B.withinyou), by the median plate with two separate sclerotised patches laterally and a longer median bulge in anterior half  and by the spermathecae separated by more than twice their diameter .Small 2022 see b, but caThe specific name refers to the type locality and is a noun in apposition.Malaysia :OP572106).TATTTGGGGCTTGAGCTTCTATAGCTGGTACATCTATAAGTGTTTTGATTCGTATGGAGTTGGGACATTCGGGGAGAATATTGGGAGATGATCATCTCTATAATGTTATTGTTACTGCTCATGCTTTTGTTATAATTTTTTTTATAGTTATACCTATTTTAATTGGAGGTTTTGGTAATTGGTTGGTTCCTTTAATGTTAGGGGCTCCTGATATGTCGTTTCCTCGAATAAATAATTTGTCTTTTTGGTTACTTCCTCCTTCTTTATTTTTATTGTTTATGTCTTCTATAACTGAAATAGGGGTAGGAGCTGGTTGAACGGTGTATCCTCCTTTGGCTTCAAGAATGGGTCATGCTGGTAGATCTATGGATTTTGCTATTTTTTCTCTTCATTTAGCTGGGGCGTCTTCTATTATAGGTGCTATTAATTTTATTTCTACTATTATTAATATGCGTTTATTAGGAATGAGAATAGAGAAAGTTCCTTTGTTTGTATGGTCTGTTTTTATTACTGCGGTATTGTTATTATTGTCTCTTCCTGTTTTGGCAGGAGCTATTACTATATTGTTAACTGATCGTAATTTTAATACTTCTTTTTTTGATCCGGCTGGAGGGGGAGATCCGATTTTATTTCAACATTTATTTTGATTTTTT : PL 6.4, PW 5.1, AW 2.3, OL 4.8, OW 3.6. Eye diameters and interdistances: AME 0.22, ALE 0.17, PME 0.27, PLE 0.23, AME–AME 0.25, AME–ALE 0.40, PME–PME 0.34, PME–PLE 0.49, AME–PME 0.17, ALE–PLE 0.29, clypeus AME 0.11, clypeus ALE 0.52. Palp and leg measurements: palp 8.2 , I 20.5 , II 17.4 , III 14.5 , IV 22.4 . Leg formula 4123. Spination of palp and legs: palp 161, 001, 111; femora I p021, d111, r1111, II p112, d111, r112, III p001, d222, r112, IV p012, d111, r112; patellae I–IV 101; tibiae I p110, d111, r11, v22222, II p110, d11, r11, v22222, III–IV p11, d111, r11, v222; metatarsi I–II p111, d012, r111, v222, III p111, d002, r111, v222, IV p111, d012, r111, v222. Chelicerae with 3 promarginal, 4 retromarginal teeth and with elongated patch of 23 tiny denticles along entire cheliceral furrow. Retromargin of chelicerae close to fang base with 6 bristles. Sparse scopula on all tarsi and metatarsi I–III. Leg claws I with 5, II with 4, III with 5 and IV with 6 secondary teeth. Position of tarsal organ: I with 1.64, II 1.15, III 0.96, IV 1.28.Palp Fig. a–c. RTA 28Colour Fig. c and d. Female (IZCAS-Ar 43733): PL 5.9, PW 4.4, AW 3.0, OL 5.2, OW 3.5. Eye diameters and interdistances: AME 0.23, ALE 0.19, PME 0.27, PLE 0.25, AME–AME 0.20, AME–ALE 0.44, PME–PME 0.33, PME–PLE 0.52, AME–PME 0.17, ALE–PLE 0.28, clypeus AME 0.13, clypeus ALE 0.51. Palp and leg measurements: palp 5.8 , I 13.9 , II 12.8 , III 11.3 , IV 16.7 . Leg formula 4123. Spination of palp and legs: palp 131, 001, 112, 203; femora I p021, d111, r011, II p012, d111, r111, III p112, d111, r112, IV p002, d111, r112; patellae I–II 000, III–IV 101; tibiae I–II v22222, III–IV p11, d111, r11, v222; metatarsi I–II v222, III–IV p111, d012, r111, v222. Chelicerae with 3 promarginal, 4 retromarginal teeth and with elongated patch of 12 tiny denticles along entire cheliceral furrow. Retromargin of chelicerae close to fang base with 7 bristles. Sparse scopula restricted almost entirely to tarsi, only metatarsi I–II with sparse scopula hairs. Palpal claw with 5 secondary teeth, leg claws I with 2, II with 3, III with 4 and IV with 3 secondary teeth. Position of tarsal organ: I 0.97, II 0.84, III 0.75, IV 1.06.Copulatory organ Fig. a and b. Colour Fig. e and f. Ctenidae . The new species is assigned to the scarymonsters-species group with the characteristics of embolus with a basal, ventral bulge , tegulum bulging proximally at tegular apophysis base, the subdistally arising, apically pointed RTA in males, transversally oval median plate and lateral teeth situated at posterior margin of epigyne and spermathecae bottle gourd-shaped. It resembles B.neukoeln Jäger, 2022 (see 28B.neukoeln), by the tegular apophysis longitudinally orientated , by the conductor nearly quadrilateral  and by the constrictive anterior width/widest width: 1/2 .Medium-sized 2022 see a–c and l2022 see a, but caThe specific name refers to the type locality and is a noun in apposition.Malaysia :OP572111).GGTTTGGAGCTTGAGCTTCTATAGTAGGAACATCTATAAGAGTATTAATTCGTATAGAATTAGGACATTCTGGAAGATTATTAGGAGATGATCATTTATATAATGTAGTTGTTACTGCTCATGCTTTTGTTATGATTTTTTTTATAGTAATGCCTATTTTAATTGGAGGTTTTGGAAATTGATTAGTTCCTTTAATATTAGGGGCTCCTGATATATCTTTTCCTCGAATAAATAATTTATCATTTTGATTACTTCCTCCTTCTTTATTCTTATTATTTATGTCTTCTATAACTGAGATAGGAGTAGGAGCTGGTTGAACAGTATATCCTCCCTTAGCTTCTAGAATAGGACATATGGGAAGATCAATGGATTTTGCTATTTTTTCTCTTCATTTAGCTGGGGCTTCCTCTATTATAGGAGCTATTAATTTTATTTCTACAATTATTAATATACGATTATTGGGAATAAGAATAGAGAAAGTTCCATTATTTGTGTGGTCTGTTTTTATTACTGCGGTATTGTTGTTATTGTCTTTACCTGTTTTAGCAGGTGCTATTACTATATTATTAACGGATCGTAATTTTAATACTTCTTTTTTTGACCCAGCTGGGGGGGGGGATCCCATTTTATTTCAACATTTATTTTGATTTTTGC :OP572109).GGTTTGGAGCTTGAGCTTCTATAGTAGGAACATCTATAAGAGTATTAATTCGTATAGAATTAGGACATTCTGGAAGATTATTAGGAGATGATCATTTATATAATGTAGTTGTTACTGCTCATGCTTTTGTTATGATTTTTTTTATAGTAATGCCTATTTTAATTGGAGGTTTTGGAAATTGATTAGTTCCTCTAATATTAGGGGCTCCTGATATATCTTTTCCTCGAATAAATAATTTATCATTTTGATTACTTCCTCCTTCTTTATTCTTATTATTTATGTCTTCTATAACTGAGATAGGAGTAGGAGCTGGTTGAACAGTATATCCTCCCTTAGCTTCTAGAATAGGACATATGGGAAGATCAATGGATTTTGCTATTTTTTCTCTTCATTTAGCTGGGGCTTCCTCTATTATAGGAGCTATTAATTTTATTTCTACAATTATTAATATACGATTATTGGGAATAAGAATAGAGAAAGTTCCATTATTTGTGTGGTCTGTTTTTATTACTGCGGTATTGTTGTTATTGTCTTTACCTGTTTTAGCAGGTGCTATTACTATATTATTAACGGATCGTAATTTTAATACTTCTTTTTTTGACCCAGCTGGGGGGGGGGATCCCATTTTATTTCAACATTTATTTTGATTTTTGC , Am.krabi sp. n. , Am.matakecil Miller & Rahmadi, 2012 , Am.phangnga sp. n.  and Am.saraburi sp. n. , the PME and AME are almost the same size  also is a cave-dwelling species and its eyes are strongly reduced in size and pigments are absent  are almost the same size (The posterior median eyes of all Li, 2022 . For theame size . These re absent . It is wame size , so thes