Cytoskeleton

The intracellular transport system is pivotal for cellular function and integrity, facilitated by cytoskeletal motor proteins such as dynein, which traverse along microtubules (MTs). The heterogeneity of the tubulin isotypes composing MTs introduces functional diversity, potentially affecting cytoskeletal motor proteins interactions with the MT. This in silico study investigated the influence of amino acid sequence variations in the C-terminal tails (CTTs) of six different Homo sapiens tubulin isotypes, TUBB2A, TUBB2B, TUBB2C, TUBB3, TUBB4A, and TUBB5, highly expressed in human brain tumors, and assessed the isotypes effect on the binding of motor protein dynein to MT. Among these isotypes, TUBB2A, TUBB2B, and TUBB2C were found to affect conformational motions of the dyneins microtubule-binding domain (MTBD) and stalk domain. The investigation highlighted the novel role of isotype-specific variations in lateral interactions between tubulin protofilaments (PFs) in determining the proximity of the {beta}-CTT of the adjacent PF to the MTBD, potentially affecting dyneins motility and suggesting how changes in isotype expression directly influence dyneins velocity and processivity and contribute to transport defects associated with neurological disorders and cancers. Isolating specific tubulin isotypes experimentally is challenging due to their high sequence similarity and complex interactions with other microtubule-associated proteins. This makes it challenging to distinguish between different tubulin isotypes and their effects, particularly in tissues where multiple isotypes are co-expressed. Additionally, these isotypes are heavily modified in vivo by post-translational modifications, which further complicate the isolation of a single, unmodified tubulin isotype. As a result, computational approaches have been necessary in this study for exploring these effects in a controlled, isotype-specific manner.

Actin-bundle organization is essential for vascular smooth muscle cell mechanics and is implicated in actin-related diseases. However, it remains unclear how cell stretching affects intracellular actin bundles when actin polymerization is impaired. Here, we performed live imaging of Latrunculin A-treated A7r5 vascular smooth muscle cells in a stretch chamber. GFP--actinin imaging showed that Latrunculin A reduced actin-bundle coverage while periodicity was maintained. Subsequent mechanical stretch disrupted both actin-bundle coverage and periodicity. We constructed a stochastic filament bundle model in which actin filament length, actin crosslinking protein dynamics, external stretch, and myosin-driven contractile shortening determine bundle connectivity. The model generated non-spanning, collapse, and persistent states based on spanning connectivity before and after stretch, shaped by filament length and applied strain. A reduced model further showed that these states are governed by a balance between connectivity formation and stretch-induced loss. Together, our results suggest that reduced actin polymerization destabilizes intracellular actin-bundle organization under mechanical stretch, providing a mechanism linking actin polymerization defects to mechanical fragility in vascular smooth muscle cells.

Cell migration depends on coordinating cell shape changes with force generation, yet how these processes are integrated remains unclear. Here, we combine live-cell imaging with traction force microscopy and computational analysis to quantify cell morphology, motility and force generation in migrating fibroblasts. We find that traction force magnitudes display a multimodal distribution, suggesting discrete migratory regimes. Using a Hidden Markov Model, we identify distinct force states that exhibit differences in shape and motion metrics, and show that individual cells transition between force states over time. To test the role of cytoskeletal organization in establishing the identified states, we analyzed cells lacking Arpc2, which disrupts branched actin assembly. Despite reduced forces and altered morphology, these cells also exhibit three migratory states. State transitions occur more frequently in cells lacking Arpc2 and unlike normal cells their protrusion geometry is force dependent. Together, our findings show that cell migration is organized into discrete mechanical states that couple morphology, motility and force generation. SUMMARY STATEMENTFibroblast motility involves distinct migratory states. These states exist independent of branched actin. However, state transition frequencies, traction force magnitudes and protrusion geometry are branched actin dependent.

Missense mutations in the TPM2 gene encoding skeletal muscle tropomyosin Tpm2.2 cause congenital myopathies associated with hyper- and hypocontractile phenotypes. Mutation-dependent defects in thin filament stability and length maintenance may contribute to sarcomere dysfunction. To address this possibility, four disease-associated substitutions in Tpm2.2 were analyzed: hypercontractile D20H and E181K, and hypocontractile E41K and N202K. Recombinant proteins were examined in vitro for their effects on actin filament polymerization, stability, and cofilin-2-dependent filament length regulation in the absence and presence of troponin (+Ca2+). Wild-type Tpm2.2 inhibited spontaneous actin polymerization and reduced polymerization cooperativity in the presence of cofilin-2. Hypercontractile substitutions D20H and E181K further decreased the polymerization rate, whereas hypocontractile variants had little effect. Under ATP-driven actomyosin interactions, E41K and N202K stabilized filaments, resulting in increased filament length, but this effect was abolished by troponin. All variants slightly decreased cofilin-2 affinity for F-actin without affecting cooperativity. Troponin prevented displacement of Tpm2.2 from the filament at increasing cofilin-2 occupancy, indicating concomitant binding of all proteins to the thin filament, consistent with a structural model based on high-resolution F-actin-Tpm-Tn and cofilactin structures.Tpm2.2-N202K inhibited cofilin-2-dependent depolymerization, whereas Tpm2.2-E181K increased susceptibility to depolymerization. Although cofilin-2 induced filament severing in all cases, the Tpm2.2-Tn complex protected filaments from disassembly. These findings support a model in which the Tpm2.2-Tn complex forms a cooperative regulatory strand that constrains filament dynamics and transmits structural perturbations along the filament. Disease-causing substitutions differentially alter filament length and stability, potentially contributing to the pathogenesis of myopathies.

Microtubules are cytoskeletal structures composed of polymers of /{beta}-tubulin heterodimers. They play a central role in cell division and motility by a stochastic process of alternating polymerization and depolymerization episodes (dynamic instability) that can be modulated by phosphorylation. Protein kinase C and cyclin-dependent kinase 1 are known to phosphorylate Ser165 of -tubulin (:Ser165) and Ser172 of {beta}-tubulin, ({beta}:Ser172), respectively. Using all-atom molecular dynamics simulations of 6-mer {beta}-tubulin systems modeled on the cryo-EM structure of a microtubule (PDB 3J6E), the impact of phosphorylation at each site is explored in terms of secondary structures (:helix H8/loop T7 segment and {beta}:loops T3/T5) that lie at the inter-dimer cleft near the E-site {beta}:GTP. If properly aligned, :Glu254 (helix H8) hydrolyzes {beta}:GTP to GDP thereby triggering the transition from a polymerizing to a depolymerizing microtubule. -Tubulin phosphorylated at :Ser165 displaces helix H8 (:Glu254/:Gln256) and loop T5 towards the {gamma}-phosphate of {beta}:GTP. This movement coincides with a shift of the {beta}:GTP nucleotide by 4.5-5.5 [A], stabilization of the {gamma}P of {beta}:GTP by additional H-bonding and weakened inter-dimer interactions. In a phosphorylated {beta}:Ser172 system, loop T5 is displaced toward {beta}:GTP and coincides with stabilization of inter-dimer interactions. Therefore, phosphorylation of either - or {beta}-tubulin generates a distinct profile of intramolecular rearrangements that remodel the inter-dimer cleft and modulate dynamic instability. These profiles may provide a useful reference for screening mutations identified in tumor genomes.

Ventricular myosin light chain-1 (MLC1v) is a key structural and function-modulating component of the {beta}-cardiac myosin ({beta}M-II) motor complex. Single-point mutations in MLC1v are linked to severe forms of hypertrophic cardiomyopathy (HCM) and sudden cardiac death (SCD) at a young age. However, the molecular mechanisms underlying the motor dysfunction responsible for HCM phenotype development are not fully understood. Here, we investigated native {beta}M-II motors isolated from septal myectomy sample of an HCM patient, harboring a rare homozygous mutation in MLC1v (A57D). Using a pure population of mutant motors (MUT), and sensitive single-molecule functional analysis approach, we directly assessed the primary functional alterations in {beta}M-II bearing A57D MLC1v mutation. In optical trap single-molecules measurements, the mutant motors displayed increased actomyosin (AM) interaction duration in strongly bound state (ton), corresponding to 3-fold reduced AM detachment rate than wild type myosin (WT). The MUT myosin also generated a shorter powerstroke size ({delta}). Ensemble average analysis of AM interaction events demonstrated that both the first powerstroke ({delta}1) associated with Pi release and the second powerstroke ({delta}2) linked to ADP release were reduced in MUT myosin. Moreover, the increased actomyosin cross-bridge stiffness in the AM.ADP state was observed for MUT compared to WT motors. Consistent with slower AM detachment rate and shorter stroke size, reconstituted human mutant {beta}M-II displayed slower actin filament gliding speed. Alterations in sarcomere-level mechanics included increased Ca2+ sensitivity of force generation and prolonged relaxation time, as predicted by FiberSim modelling. Molecular dynamics simulations indicated that the substitution of alanine by aspartate altered MLC1v interactions with myosin heavy chain (MyHC) and light chain 2 (MLC2v), affecting the curvature and flexibility of the lever arm. Overall, these studies establish the molecular mechanism underlying the primary myosin dysfunction due to A57D MLC1v mutation and further highlight the crucial role of MLC1v-mediated regulation of myosin function. Understanding the precise changes in the mutant myosins biomechanical properties is directly relevant to comprehending the initial triggers for pathological cardiac remodeling in HCM patients and designing tailored therapeutic interventions.

A hallmark of damaged skeletal muscle fibers is displaced myonuclei that are no longer peripherally positioned. Displaced myonuclei are dogmatically thought to be derived exclusively from muscle stem cell (satellite cell) fusion. Using a surgical resection muscle injury model and in vivo recombination-independent resident myonuclear labeling, we detail the prevalence, time course, and origin of displaced myonuclei in response to a non-chemically-mediated muscle trauma. We found that: 1) non-satellite cell-derived (resident) displaced myonuclei emerge seven days after surgical injury in similar proportion to exogenous (satellite cell-derived) displaced myonuclei in intact muscle fibers, with a biased prevalence in myosin heavy chain IIB muscle fibers, 2) muscle fibers with multiple ([≥]2) displaced resident myonuclei was an unexpected but noteworthy feature of muscle fibers 7 days after injury, 3) embryonic myosin-expressing fibers at seven days post-surgery expectedly contain predominantly satellite-cell derived displaced myonuclei, but a subset have displaced resident myonuclei, and 4) satellite cell numbers in intact muscle do not increase until 7 days post-surgery. These data may help inform whether to target satellite cell-initiated processes, myonuclear-initiated processes, or both to facilitate muscle fiber injury repair. This information could lead to more effective therapeutic strategies for treating muscle trauma.

Mechanosensing involves proteins detecting mechanical changes in the cytoskeleton or at cell adhesion sites. These interactions initiate signaling cascades that produce biochemical effects such as post-translational modifications or cytoskeletal rearrangements. Filamin is a ubiquitous mechanosensing protein that binds actin filaments and senses pulling forces within the cytoskeleton. Drosophila Filamin (Cheerio) is structurally similar to mammalian Filamin, with roles in egg chamber development, embryo cellularization, and integrity of muscle attachment sites and Z discs in Drosophila indirect flight muscles (IFMs). Here we report a potential novel binding partner of Drosophila Filamins: the death-associated protein kinase Drak that functions as a myosin light chain kinase. We found that Drak biochemically bound to an open mutant of Filamin that resembles the mechanically activated form partially bound to wild type Filamin and did not bind to closed mutant of Filamin. The interaction site was mapped to the intrinsically unfolded C-terminal region of Drak. To study the functional role of Drak-Filamin interaction, we studied two developmental events where Drak has been earlier shown to be expressed and where Filamin also functions: early embryonic cellularization and indirect flight muscle development at pupal stages. We found partial colocalization between Drak-GFP and Filamin-mCherry during the initiation of cellularization furrow, and at the time of myotube attachment site maturation in tendon cells. However, functionally we could not show direct correlation between Filamin and Drak. Our studies reveal interesting new expression patterns of Drak during Drosophila development and provide detailed information about Filamin localization during IFM development.

The Drosophila scaffolding protein Zasp52 is required to maintain structure at the muscle Z-disc which experiences strong forces during contraction. It is alternatively spliced into many isoforms, some of which contain a long intrinsically disordered region (IDR). We show that this region is primarily expressed in the indirect flight muscle (IFM) and is required for maintaining integrity of the Z-disc. Deleting the IDR-encoding exon15e results in flightlessness and structural IFM defects, including sarcomere bending at the Z-disc and an inability to de-contract. These defects are indicative of a lack of proper thin filament anchoring to the Z-disc. This is further supported by a genetic interaction between exon15e and actin. Fluorescence recovery after photobleaching of an isoform lacking exon15e shows that the IDR is required for maintaining Zasp52 at the Z-disc and thereby stabilizing Z-discs. Lastly, we can rescue these phenotypes by restricting IFM use. Together, these results suggest that Zasp52s IDR confers thin filament stability at the Z-disc of IFM.

Motile cells can sense and exert forces on the extracellular environment through dynamic actin networks. Increased stress against the polymerizing barbed ends of branched actin networks has been shown to lead to an increase in the density of these networks through a force feedback mechanism, though this phenomenon has not been explored through the examination of real-time responses of endogenous actin networks in cells. Here, we utilize mouse embryonic fibroblast CRISPR knock-in lines with labeled ARP2/3 complex to identify cellular and extracellular conditions that regulate branched actin density and enrichment at the leading edge of lamellipodial protrusions. A common theme shared among all branched actin density-increasing conditions is higher levels of interface stress between the plasma membrane and the barbed ends of the lamellipodial actin network. Among these conditions, we find that ARP2/3 is specifically required for robust spreading and protrusion in response to increased extracellular viscosity. Interestingly, time-lapse traction force microscopy of ARP2/3-dependent viscosity responses show significantly reduced changes in strain energy applied to the substrate when compared to spreading and motility through cell-matrix adhesion. In addition, we find that increased extracellular viscosity can bypass the need for extracellular matrix proteins to support lamellipodial protrusion driven by optogenetic Rac activation. Our studies provide strong support for in vitro models of branched actin force feedback responses and further characterize an essential role for branched actin in mediating dramatic cell shape changes in response to increased extracellular viscosity.

Increased mechanical loading induces skeletal muscle growth and, at the ultrastructural level, promotes myofibrillogenesis and the radial growth of myofibrils. However, the mechanisms regulating these ultrastructural adaptations are not known. Here, we sought to determine whether the mechanistic target of rapamycin complex 1 (mTORC1) regulates these processes. To accomplish this, muscle-specific, tamoxifen-inducible raptor knockout (iRAmKO) mice were used to inhibit signaling through mTORC1, and growth was induced with a model of chronic mechanical overload (MOV). Using a next-generation fluorescence imaging pipeline for ultrastructural analyses, we found that mTORC1 is a critical regulator of the myofibrillogenesis and radial growth of myofibrils that occur in response to MOV. Together with other recent advances in the field, we propose a model in which mTORC1 acts as a gatekeeper that permits the retention, rather than the synthesis, of proteins that drive the ultrastructural adaptations.

Cell contractility plays numerous essential roles in a healthy organism, while its malfunctioning can lead to disease. The ubiquitous actin-dependent motors of the nonmuscle myosin 2 (NM2) family, which includes NM2A and NM2B, are chiefly responsible for cell contractility because of their ability to polymerize into bipolar filaments. Polymerization/depolymerization of NM2 filaments allows cells to quickly reorganize their contractile system according to constantly changing shapes and positions of nonmuscle cells. Bipolar filament depolymerization is known to depend on the C-terminal features of the NM2A heavy chain. Here, we show that the motor activity of NM2A is another key component of NM2As depolymerization mechanism, which cooperates with tail-dependent mechanisms to facilitate NM2A turnover in cells and, through copolymerization with NM2B, to reorganize and dynamize NM2B in trans, thus generating a proper intracellular NM2A/NM2B distribution needed for efficient cell migration. Together, we show that NM2A motor activity is a key component of the bipolar filament depolymerization mechanism.

The structural integrity of spinal cord tissue and the transmission of mechanical stimuli across the different levels of tissue microarchitecture and varying spatial scales of mechanical loading challenge experimental and computational efforts to accurately model, simulate and interpret tissue mechanics, leading to conflicting findings in existing literature. Here, we demonstrate that the bead size used in spherical indentation tests significantly affects the stiffness ratio of spinal cord gray to white matter, a dependence which we only observe on the transverse plane and not the coronal plane of the tissue. Our study reveals a shift in stiffness ratio such that for smaller spherical indenters gray matter is stiffer than white matter, while for larger indenters, white matter is stiffer than gray matter. The mean relative change from the 100 {micro}m bead to the 500 {micro}m bead differed between anatomical planes, with transverse sections showing a decrease in gray matter (-13.3%) and an increase in white matter stiffness (+26.9%), accompanied by a reduction in the gray-to-white matter stiffness ratio from 1.07 to 0.76, whereas coronal sections exhibited increases in both gray (+21.0%) and white matter (+33.8%), along with a change in the ratio from 0.99 to 1.14. These findings contribute to explaining previously contradictory results in the literature and underscore the relevance of spatial scales in mechanical characterization studies.

Huntingtons disease is caused by expansion of a CAG repeat in the human HTT gene, producing a mutant huntingtin protein that misfolds and forms intracellular aggregates. Although Huntingtons disease is primarily characterized as a neurodegenerative disorder, mutant huntingtin is ubiquitously expressed, and peripheral tissues such as skeletal muscle exhibit pathological abnormalities. To define the muscle-intrinsic consequences of pathogenic huntingtin expression, we expressed caspase-6 truncated pathogenic human huntingtin in body wall muscle of Drosophila melanogaster larvae and performed quantitative structural and functional analyses. Aggregate analysis revealed that fluorescence intensity increased with aggregate size while aggregate morphology became more irregular. Delaying transgene expression until later stages of larval development dramatically reduced aggregate number, demonstrating a strong temporal dependence of aggregate formation. Myonuclei were enlarged, misshapen, and exhibited significantly reduced fluorescence intensity, consistent with altered chromatin organization. Notably, huntingtin aggregates were observed within the nucleus, indicating that nuclear proteostasis is directly perturbed by pathogenic huntingtin in muscle cells. Despite these intracellular defects, muscle fiber shape and sarcomere organization were preserved, suggesting that contractile apparatus assembly is not overtly disrupted. In contrast, mitochondrial organization was severely affected, with extensive mitochondrial aggregation throughout muscle fibers, consistent with altered organelle homeostasis. Functional analyses demonstrated that pathogenic huntingtin expression significantly impaired neuromuscular performance. Larvae exhibited reduced excitatory junctional potentials and diminished muscle contractile force, indicating compromised synaptic transmission and muscle function. Together, these findings demonstrate that pathogenic human huntingtin expression in skeletal muscle is sufficient to drive widespread protein aggregation, nuclear and mitochondrial abnormalities, and functional deficits despite the absence of overt structural changes. Our results highlight the importance of muscle-intrinsic pathogenic mechanisms and provide a quantitative framework for understanding how mutant huntingtin disrupts cellular organization and physiology outside the nervous system.

1Nuclear envelope stretch and rupture are common to cell spreading and migration in a variety of microenvironments, leading to marked changes in nucleocytoplasmic transport. Predicting cell response to different mechanochemical cues that are transmitted to the nucleus remains an open problem in the field of mechanomedicine. We developed a predictive modeling framework to examine how nuclear deformation on substrates with different nanotopographies influences nucleocytoplasmic transport and rearrangement of the nuclear lamina. Using the finite element method, we simulated nuclear compression by the perinuclear actin cap on substrates with arrays of nanopillars, modeling the nuclear envelope as a nonlinear elastic structure and coupling deformations to a biochemical model of lamin remodeling and nucleocytoplasmic transport. These simulations predicted regions of high nuclear envelope stretch adjacent to cell-nanopillar contacts, leading to maximized nuclear envelope tension on small nanopillars spaced by 4-5 microns. We then considered the effects on nuclear transport of YAP and TAZ and found that increased nuclear compression led to YAP/TAZ nuclear localization in agreement with previous experiments. Furthermore, the simulated force load per lamin was maximized on nanopillar substrates with high nuclear stretch. The magnitude of this load was modulated by the rate of actin cap assembly and the overall expression level of lamin A/C - decreasing lamin content in the nuclear envelope led to a higher likelihood of rupture. We validated this prediction in subsequent experiments with lamin-depleted U2OS cells, establishing the central importance of lamin transport and microenvironment nanotopography to nuclear mechanotransduction. 2 SignificanceCell nuclei commonly experience large strains, but existing computational models do not explain the coupling between such deformations and molecular transport. Here, we present a modeling framework that includes the mechanics of nuclear deformations and the reaction-transport of molecules within the cytoplasm, nuclear envelope, and nuclear interior. As a well-controlled setup for comparing experiments and simulations, we consider nuclear indentations exhibited by cells on nanopillar substrates. Our simulations recapitulate measurements of nuclear YAP/TAZ localization from the literature and predict that low-lamin cells experience higher force loads at the nuclear envelope. We validate this prediction experimentally, showing that lamin-depleted cells are more likely to exhibit nuclear rupture. Overall, our framework presents opportunities to predict nuclear mechanoadaptation to different microenvironments.

Cofilin is a key regulator of actin dynamics that, along with a myriad of other actin-binding proteins, controls the balance of F- and G-actin in numerous cell types. While prior structural studies of the cofilin-actin binding interface have delineated many critical interactions between cofilin and actin, the roles of some residues within the cofilin-actin binding interface remain poorly defined. In this study, we investigate the role of cofilin S119 in the cofilin-actin interaction. Despite its unique position within the cofilin-actin interface and its putative role as a phosphorylation site, relatively little direct evidence exists to define whether it plays an important role in cofilin-actin dynamics. Using site-directed mutagenesis, we demonstrate that mutation of S119 to aromatic amino acids (W, F, Y) results in cofilins with strong actin bundling activity in living cells. This activity can be countered by the incorporation of mutants that disfavor actin rod forming activity (R21Q). Mutation of S119 to phospho-mimic (E) and non-phosphorylated (A) residues either strongly inhibits (E) or modestly increases (A) actin bundling activity. Expression of the S119W mutant in neurons reveals its impacts on spine length and size, while FRAP studies show that its mobile fraction is intermediate between that of LifeAct and WT cofilin. Finally, it is shown that the strong actin bundling phenotype associated with S119W inhibits the progression of optogenetically induced apoptosis.

Cells are mechanosensitive, responding to external mechanical stimulation. Nanovibrational stimulation has been shown to enhance cell contractility and actin stress fibre formation. These changes in morphology occur quickly, alongside associated mechanical changes. Here, the relationship between acute morphological and mechanical changes in NIH 3T3 fibroblastic cells in response to nanovibrational stimulation is presented. A 1 kHz, 30 nm vibration is applied continuously for 72 hours. Atomic force microscopy (AFM) quantifies mechanical properties of the nucleus and cytoplasm at multiple timepoints, while immunofluorescence tracks morphological changes. Within 3 hours of stimulation, both nuclear and cytoplasmic stiffness increase significantly, accompanied by a decrease in the cellular fluid exponent, suggesting a shift of the cell towards more solid-like behaviour. These changes correlate with increased nuclear area. Actin polymerisation also increases within 24 hours, although variably. To understand the role of the cytoskeleton, actin polymerisation and contraction are inhibited using cytochalasin D and blebbistatin. Results show that inhibition prevents stiffness increases and results in a higher fluid exponent, indicating a more fluid-like state. These findings demonstrate that actin-myosin dynamics mediate cell stiffening under nanovibrational stimulation. Interestingly, prolonged stimulation appears to reverse this effect, suggesting that temporal optimisation of stimulation may enhance long-term mechanotransducive responses.

Microtubule networks are major determinants of cell architecture and logistics. Microtubule organization and density are regulated by severing enzymes, which cut microtubule lattices or affect their growth and shortening. These activities can lead to microtubule amplification or disassembly, depending on the presence of microtubule stabilizers or destabilizers, but the interplay between these factors is poorly understood. Here, we reconstituted in vitro the activity of microtubule severase katanin together with microtubule minus-end stabilizers CAMSAPs, their binding partner WDR47 and microtubule depolymerase kinesin-13/MCAK. We confirmed that katanin can amplify or destroy microtubules in a concentration-dependent manner. CAMSAPs recruit katanin to microtubules and reduce katanin concentration needed for both amplification and destruction, whereas kinesin-13 completely abolishes microtubule amplification. WDR47 binds to microtubules decorated by CAMSAPs and suppresses katanin binding and severing. In addition, both katanin and WDR47 inhibit polymerization of CAMSAP-decorated microtubule minus ends. These data explain how these proteins act together to fine-tune microtubule minus-end stability without strongly increasing microtubule abundance. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=169 SRC="FIGDIR/small/714132v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@746fe3org.highwire.dtl.DTLVardef@5dd5a8org.highwire.dtl.DTLVardef@762373org.highwire.dtl.DTLVardef@1192db_HPS_FORMAT_FIGEXP M_FIG Graphical abstract C_FIG

Cell adhesion enables animal multicellular development. E-cadherin and the cadherin-catenin adhesion complex at adherens junctions are engaged in dynamic interactions with actomyosin generated contractile forces to drive epithelial morphogenesis. However, our understanding of how adhesion is regulated and how the tuning of adhesion contributes to morphogenesis remains incomplete. One key determinant of E-cadherin adhesion strength is clustering of the cadherin-catenin adhesion complex, a property studied extensively in vitro. Here, we use optogenetics to enhance E-cadherin cluster formation in the Drosophila embryo. Enlarged clusters were associated with increased E-cadherin surface abundance, assembled a normal cadherin-catenin complex, and showed reduced membrane mobility and turnover consistent with an increase in cell adhesion strength. Drosophila embryos with enhanced E-cadherin clustering displayed a severe reduction in cell intercalation and convergent extension of the anterior-posterior axis. To account for these observations, we modified existing vertex models to include junction-specific viscous forces representing E-cadherin-mediated friction between cells. This dissipative adhesion model predicts that enhanced adhesion increases resistance to cell rearrangements, thereby reducing cell neighbor exchanges and impairing convergent extension. To test model predictions, we analyzed two types of morphogenetic movements in embryos with enhanced E-cadherin clustering. Neuroblast ingression, which requires both apical constriction and cell rearrangement, was severely slowed. In contrast, mesoderm invagination, which requires apical constriction without neighbor exchanges, proceeded normally. Our findings suggest that optogenetic clustering, in contrast to overexpression of E-cadherin, is a valuable tool to examine the consequences of enhancing adhesion strength in tissue morphogenesis. Moreover, we propose that regulating E-cadherin clustering is essential for movements that require cell-cell contact changes.

GFAP is a type III intermediate filament primarily found within astrocytes and is known to maintain proper cell structure and mechanical strength. Mutations in GFAP are implicated in the pathology of Alexander disease, a neurodegenerative disease characterized by cytoplasmic inclusions of protein, known as Rosenthal fibers. GFAP has a typical type III intermediate filament domain structure, consisting of a highly conserved alpha-helical rod domain bracketed by an intrinsically disordered N-terminal head and C-terminal tail domains. While the general domain organization of monomeric GFAP and the assembly process for higher order quaternary structures are known, we lack an atomic resolution mechanistic understanding of GFAP assembly into mature filaments. Understanding the structure of GFAP filaments and how mutations disrupt this structure will provide vital information into how mutations produce Alexander disease pathology. As a first step towards a mechanistic description, we characterized GFAP wild type tetrameric and filamentous assemblies using solid state NMR and compared the results to those obtained from an assembly-deficient GFAP mutant. For wild-type GFAP, we observe surprisingly uniform rigid alpha helical structure and can spectroscopically resolve highly mobile intrinsically disordered regions in the filament assemblies. Wild type tetramers show increased mobility, likely arising from the head and tail domains. Mutation of the highly conserved cysteine at position 294 to serine results in an inability to form full-length filament assemblies. We show that the rigid regions of the C294S mutant assemblies largely remain structurally consistent with wild type tetrameric assemblies but differ from wild-type filament assemblies. There is an increase in highly mobile regions for the C294S mutant relative to the wild-type. Our results provide a foundation for developing solid state NMR approaches to characterize intermediate filament assembly mechanisms and the interfering effect of disease mutations.