Vitamin C Drives Reentrant Actin Phase Transition: Biphasic Exocytosis Regulation Revealed by Single-Vesicle Electrochemistry.

Unveiling molecular mechanisms that dominate protein phase dynamics has been a pressing need for deciphering the intricate intracellular modulation machinery. While ions and biomacromolecules have been widely recognized for modulating protein phase separations, effects of small molecules that essentially constitute the cytosolic chemical atmosphere on the protein phase behaviors are rarely understood. Herein, we report that vitamin C (VC), a key small molecule for maintaining a reductive intracellular atmosphere, drives reentrant phase transitions of myosin II/F-actin (actomyosin) cytoskeletons. The actomyosin bundle condensates dissemble in the low-VC regime and assemble in the high-VC regime in vitro or inside neuronal cells, through a concurrent myosin II protein aggregation-dissociation process with monotonic VC concentration increase. Based on this finding, we employ in situ single-cell and single-vesicle electrochemistry to demonstrate the quantitative modulation of catecholamine transmitter vesicle exocytosis by intracellular VC atmosphere, i.e., exocytotic release amount increases in the low-VC regime and decreases in the high-VC regime. Furthermore, we show how VC regulates cytomembrane-vesicle fusion pore dynamics through counteractive or synergistic effects of actomyosin phase transitions and the intracellular free calcium level on membrane tensions. Our work uncovers the small molecule-based reversive protein phase regulatory mechanism, paving a new way to chemical neuromodulation and therapeutic repertoire expansion.

LUZP1 regulates the maturation of contractile actomyosin bundles.

Contractile actomyosin bundles play crucial roles in various physiological processes, including cell migration, morphogenesis, and muscle contraction. The intricate assembly of actomyosin bundles involves the precise alignment and fusion of myosin II filaments, yet the underlying mechanisms and factors involved in these processes remain elusive. Our study reveals that LUZP1 plays a central role in orchestrating the maturation of thick actomyosin bundles. Loss of LUZP1 caused abnormal cell morphogenesis, migration, and the ability to exert forces on the environment. Importantly, knockout of LUZP1 results in significant defects in the concatenation and persistent association of myosin II filaments, severely impairing the assembly of myosin II stacks. The disruption of these processes in LUZP1 knockout cells provides mechanistic insights into the defective assembly of thick ventral stress fibers and the associated cellular contractility abnormalities. Overall, these results significantly contribute to our understanding of the molecular mechanism involved in actomyosin bundle formation and highlight the essential role of LUZP1 in this process.

The DIAPH3 linker specifies a ß-actin network that maintains RhoA and Myosin-II at the cytokinetic furrow.

Cytokinesis is the final step of the cell division cycle that leads to the formation of two new cells. Successful cytokinesis requires significant remodelling of the plasma membrane by spatially distinct ß- and γ-actin networks. These networks are generated by the formin family of actin nucleators, DIAPH3 and DIAPH1 respectively. Here we show that ß- and γ-actin perform specialized and non-redundant roles in cytokinesis and cannot substitute for one another. Expression of hybrid DIAPH1 and DIAPH3 proteins with altered actin isoform specificity relocalized cytokinetic actin isoform networks within the cell, causing cytokinetic failure. Consistent with this we show that ß-actin networks, but not γ-actin networks, are required for the maintenance of non-muscle myosin II and RhoA at the cytokinetic furrow. These data suggest that independent and spatially distinct actin isoform networks form scaffolds of unique interactors that facilitate localized biochemical activities to ensure successful cell division.

Myosin II mediates Shh signals to shape dental epithelia via control of cell adhesion and movement.

The development of ectodermal organs begins with the formation of a stratified epithelial placode that progressively invaginates into the underlying mesenchyme as the organ takes its shape. Signaling by secreted molecules is critical for epithelial morphogenesis, but how that information leads to cell rearrangement and tissue shape changes remains an open question. Using the mouse dentition as a model, we first establish that non-muscle myosin II is essential for dental epithelial invagination and show that it functions by promoting cell-cell adhesion and persistent convergent cell movements in the suprabasal layer. Shh signaling controls these processes by inducing myosin II activation via AKT. Pharmacological induction of AKT and myosin II can also rescue defects caused by the inhibition of Shh. Together, our results support a model in which the Shh signal is transmitted through myosin II to power effective cellular rearrangement for proper dental epithelial invagination.

Cell-size-dependent regulation of Ezrin dictates epithelial resilience to stretch by countering myosin-II-mediated contractility.

The epithelial adaptations to mechanical stress are facilitated by molecular and tissue-scale changes that include the strengthening of junctions, cytoskeletal reorganization, and cell-proliferation-mediated changes in tissue rheology. However, the role of cell size in controlling these properties remains underexplored. Our experiments in the zebrafish embryonic epidermis, guided by theoretical estimations, reveal a link between epithelial mechanics and cell size, demonstrating that an increase in cell size compromises the tissue fracture strength and compliance. We show that an increase in E-cadherin levels in the proliferation-deficient epidermis restores epidermal compliance but not the fracture strength, which is largely regulated by Ezrin-an apical membrane-cytoskeleton crosslinker. We show that Ezrin fortifies the epithelium in a cell-size-dependent manner by countering non-muscle myosin-II-mediated contractility. This work uncovers the importance of cell size maintenance in regulating the mechanical properties of the epithelium and fostering protection against future mechanical stresses.

S100A4 makes two appearances in mechanisms leading to fibrosis.

Non-muscle myosin 2 (NM2) is known to play an important role in myofibroblast transdifferentiation, a hallmark of fibrotic disorders. In a recent JBC article, Southern et al. demonstrate that endogenous S100A4, a calcium- and NM2-binding protein acts as a mechanoeffector in this process. Since extracellular S100A4 is also involved in fibrogenesis by triggering the inflammatory response, this small protein appears to contribute to fibrosis via at least two distinct mechanisms.

Focal adhesions are controlled by microtubules through local contractility regulation.

Microtubules regulate cell polarity and migration via local activation of focal adhesion turnover, but the mechanism of this process is insufficiently understood. Molecular complexes containing KANK family proteins connect microtubules with talin, the major component of focal adhesions. Here, local optogenetic activation of KANK1-mediated microtubule/talin linkage promoted microtubule targeting to an individual focal adhesion and subsequent withdrawal, resulting in focal adhesion centripetal sliding and rapid disassembly. This sliding is preceded by a local increase of traction force due to accumulation of myosin-II and actin in the proximity of the focal adhesion. Knockdown of the Rho activator GEF-H1 prevented development of traction force and abolished sliding and disassembly of focal adhesions upon KANK1 activation. Other players participating in microtubule-driven, KANK-dependent focal adhesion disassembly include kinases ROCK, PAK, and FAK, as well as microtubules/focal adhesion-associated proteins kinesin-1, APC, and αTAT. Based on these data, we develop a mathematical model for a microtubule-driven focal adhesion disruption involving local GEF-H1/RhoA/ROCK-dependent activation of contractility, which is consistent with experimental data.

Patterning and folding of intestinal villi by active mesenchymal dewetting.

Tissue folds are structural motifs critical to organ function. In the intestine, bending of a flat epithelium into a periodic pattern of folds gives rise to villi, finger-like protrusions that enable nutrient absorption. However, the molecular and mechanical processes driving villus morphogenesis remain unclear. Here, we identify an active mechanical mechanism that simultaneously patterns and folds the intestinal epithelium to initiate villus formation. At the cellular level, we find that PDGFRA+ subepithelial mesenchymal cells generate myosin II-dependent forces sufficient to produce patterned curvature in neighboring tissue interfaces. This symmetry-breaking process requires altered cell and extracellular matrix interactions that are enabled by matrix metalloproteinase-mediated tissue fluidization. Computational models, together with in vitro and in vivo experiments, revealed that these cellular features manifest at the tissue level as differences in interfacial tensions that promote mesenchymal aggregation and interface bending through a process analogous to the active dewetting of a thin liquid film.

Mechanical stress combines with planar polarised patterning during metaphase to orient embryonic epithelial cell divisions.

The planar orientation of cell division (OCD) is important for epithelial morphogenesis and homeostasis. Here, we ask how mechanics and antero-posterior (AP) patterning combine to influence the first divisions after gastrulation in the Drosophila embryonic epithelium. We analyse hundreds of cell divisions and show that stress anisotropy, notably from compressive forces, can reorient division directly in metaphase. Stress anisotropy influences the OCD by imposing metaphase cell elongation, despite mitotic rounding, and overrides interphase cell elongation. In strongly elongated cells, the mitotic spindle adapts its length to, and hence its orientation is constrained by, the cell long axis. Alongside mechanical cues, we find a tissue-wide bias of the mitotic spindle orientation towards AP-patterned planar polarised Myosin-II. This spindle bias is lost in an AP-patterning mutant. Thus, a patterning-induced mitotic spindle orientation bias overrides mechanical cues in mildly elongated cells, whereas in strongly elongated cells the spindle is constrained close to the high stress axis.

Environmentally dependent and independent control of 3D cell shape.

How cancer cells determine their shape in response to three-dimensional (3D) geometric and mechanical cues is unclear. We develop an approach to quantify the 3D cell shape of over 60,000 melanoma cells in collagen hydrogels using high-throughput stage-scanning oblique plane microscopy (ssOPM). We identify stereotypic and environmentally dependent changes in shape and protrusivity depending on whether a cell is proximal to a flat and rigid surface or is embedded in a soft environment. Environmental sensitivity metrics calculated for small molecules and gene knockdowns identify interactions between the environment and cellular factors that are important for morphogenesis. We show that the Rho guanine nucleotide exchange factor (RhoGEF) TIAM2 contributes to shape determination in environmentally independent ways but that non-muscle myosin II, microtubules, and the RhoGEF FARP1 regulate shape in ways dependent on the microenvironment. Thus, changes in cancer cell shape in response to 3D geometric and mechanical cues are modulated in both an environmentally dependent and independent fashion.

F-actin architecture determines the conversion of chemical energy into mechanical work.

Mechanical work serves as the foundation for dynamic cellular processes, ranging from cell division to migration. A fundamental driver of cellular mechanical work is the actin cytoskeleton, composed of filamentous actin (F-actin) and myosin motors, where force generation relies on adenosine triphosphate (ATP) hydrolysis. F-actin architectures, whether bundled by crosslinkers or branched via nucleators, have emerged as pivotal regulators of myosin II force generation. However, it remains unclear how distinct F-actin architectures impact the conversion of chemical energy to mechanical work. Here, we employ in vitro reconstitution of distinct F-actin architectures with purified components to investigate their influence on myosin ATP hydrolysis (consumption). We find that F-actin bundles composed of mixed polarity F-actin hinder network contraction compared to non-crosslinked network and dramatically decelerate ATP consumption rates. Conversely, linear-nucleated networks allow network contraction despite reducing ATP consumption rates. Surprisingly, branched-nucleated networks facilitate high ATP consumption without significant network contraction, suggesting that the branched network dissipates energy without performing work. This study establishes a link between F-actin architecture and myosin energy consumption, elucidating the energetic principles underlying F-actin structure formation and the performance of mechanical work.

Polarised cell intercalation during Drosophila axis extension is robust to an orthogonal pull by the invaginating mesoderm.

As tissues grow and change shape during animal development, they physically pull and push on each other, and these mechanical interactions can be important for morphogenesis. During Drosophila gastrulation, mesoderm invagination temporally overlaps with the convergence and extension of the ectodermal germband; the latter is caused primarily by Myosin II-driven polarised cell intercalation. Here, we investigate the impact of mesoderm invagination on ectoderm extension, examining possible mechanical and mechanotransductive effects on Myosin II recruitment and polarised cell intercalation. We find that the germband ectoderm is deformed by the mesoderm pulling in the orthogonal direction to germband extension (GBE), showing mechanical coupling between these tissues. However, we do not find a significant change in Myosin II planar polarisation in response to mesoderm invagination, nor in the rate of junction shrinkage leading to neighbour exchange events. We conclude that the main cellular mechanism of axis extension, polarised cell intercalation, is robust to the mesoderm invagination pull. We find, however, that mesoderm invagination slows down the rate of anterior-posterior cell elongation that contributes to axis extension, counteracting the tension from the endoderm invagination, which pulls along the direction of GBE.

Regulation of Notch signaling by non-muscle myosin II Zipper in Drosophila.

The Notch pathway is an evolutionarily conserved signaling system that is intricately regulated at multiple levels and it influences different aspects of development. In an effort to identify novel components involved in Notch signaling and its regulation, we carried out protein interaction screens which identified non-muscle myosin II Zipper (Zip) as an interacting partner of Notch. Physical interaction between Notch and Zip was further validated by co-immunoprecipitation studies. Immunocytochemical analyses revealed that Notch and Zip co-localize within same cytoplasmic compartment. Different alleles of zip also showed strong genetic interactions with Notch pathway components. Downregulation of Zip resulted in wing phenotypes that were reminiscent of Notch loss-of-function phenotypes and a perturbed expression of Notch downstream targets, Cut and Deadpan. Further, synergistic interaction between Notch and Zip resulted in highly ectopic expression of these Notch targets. Activated Notch-induced tumorous phenotype of larval tissues was enhanced by over-expression of Zip. Notch-Zip synergy resulted in the activation of JNK pathway that consequently lead to MMP activation and proliferation. Taken together, our results suggest that Zip may play an important role in regulation of Notch signaling.

Engines of change: Nonmuscle myosin II in mechanobiology.

The emergence of mechanobiology has unveiled complex mechanisms by which cells adjust intracellular force production to their needs. Most communicable intracellular forces are generated by myosin II, an actin-associated molecular motor that transforms adenosine triphosphate (ATP) hydrolysis into contraction in nonmuscle and muscle cells. Myosin II-dependent force generation is tightly regulated, and deregulation is associated with specific pathologies. Here, we focus on the role of myosin II (nonmuscle myosin II, NMII) in force generation and mechanobiology. We outline the regulation and molecular mechanism of force generation by NMII, focusing on the actual outcome of contraction, that is, force application to trigger mechanosensitive events or the building of dissipative structures. We describe how myosin II-generated forces drive two major types of events: modification of the cellular morphology and/or triggering of genetic programs, which enhance the ability of cells to adapt to, or modify, their microenvironment. Finally, we address whether targeting myosin II to impair or potentiate its activity at the motor level is a viable therapeutic strategy, as illustrated by recent examples aimed at modulating cardiac myosin II function in heart disease.

Drosophila Fog/Cta and T48 pathways have overlapping and distinct contributions to mesoderm invagination.

The regulation of the cytoskeleton by multiple signaling pathways, sometimes in parallel, is a common principle of morphogenesis. A classic example of regulation by parallel pathways is Drosophila gastrulation, where the inputs from the Folded gastrulation (Fog)/Concertina (Cta) and the T48 pathways induce apical constriction and mesoderm invagination. Whether there are distinct roles for these separate pathways in regulating the complex spatial and temporal patterns of cytoskeletal activity that accompany early embryo development is still poorly understood. We investigated the roles of the Fog/Cta and T48 pathways and found that, by themselves, the Cta and T48 pathways both promote timely mesoderm invagination and apical myosin II accumulation, with Cta being required for timely cell shape change ahead of mitotic cell division. We also identified distinct functions of T48 and Cta in regulating cellularization and the uniformity of the apical myosin II network, respectively. Our results demonstrate that both redundant and distinct functions for the Fog/Cta and T48 pathways exist.

Local monomer levels and established filaments potentiate non-muscle myosin 2 assembly.

The ability to dynamically assemble contractile networks is required throughout cell physiology, yet direct biophysical mechanisms regulating non-muscle myosin 2 filament assembly in living cells are lacking. Here, we use a suite of dynamic, quantitative imaging approaches to identify deterministic factors that drive myosin filament appearance and amplification. We find that actin dynamics regulate myosin assembly, but that the static actin architecture plays a less clear role. Instead, remodeling of actin networks modulates the local myosin monomer levels and facilitates assembly through myosin:myosin-driven interactions. Using optogenetically controlled myosin, we demonstrate that locally concentrating myosin is sufficient to both form filaments and jump-start filament amplification and partitioning. By counting myosin monomers within filaments, we demonstrate a myosin-facilitated assembly process that establishes filament stacks prior to partitioning into clusters that feed higher-order networks. Together, these findings establish the biophysical mechanisms regulating the assembly of non-muscle contractile structures that are ubiquitous throughout cell biology.

Suggesting Dictyostelium as a Model for Disease-Related Protein Studies through Myosin II Polymerization Pathway.

Dictyostelium myosin II displays remarkable dynamism within the cell, continually undergoing polymerization and depolymerization processes. Under low-ion conditions, it assumes a folded structure like muscle myosins and forms thick filaments through polymerization. In our study, we presented intermediate structures observed during the early stages of polymerization of purified myosin via negative staining electron microscopy, immediately crosslinked with glutaraldehyde at the onset of polymerization. We identified folded monomers, dimers, and tetramers in the process. Our findings suggest that Dictyostelium myosin II follows a polymerization pathway in vitro akin to muscle myosin, with folded monomers forming folded parallel and antiparallel dimers that subsequently associate to create folded tetramers. These folded tetramers eventually unfold and associate with other tetramers to produce long filaments. Furthermore, our research revealed that ATP influences filament size, reducing it regardless of the status of RLC phosphorylation while significantly increasing the critical polymerization concentrations from 0.2 to 9 nM. In addition, we demonstrate the morphology of fully matured Dictyostelium myosin II filaments.

Identification of novel MYH14 variants in families with autosomal dominant sensorineural hearing loss.

Autosomal dominant sensorineural hearing loss (ADSNHL) is a genetically heterogeneous disorder caused by pathogenic variants in various genes, including MYH14. However, the interpretation of pathogenicity for MYH14 variants remains a challenge due to incomplete penetrance and the lack of functional studies and large families. In this study, we performed exome sequencing in six unrelated families with ADSNHL and identified five MYH14 variants, including three novel variants. Two of the novel variants, c.571G > C (p.Asp191His) and c.571G > A (p.Asp191Asn), were classified as likely pathogenic using ACMG and Hearing Loss Expert panel guidelines. In silico modeling demonstrated that these variants, along with p.Gly1794Arg, can alter protein stability and interactions among neighboring molecules. Our findings suggest that MYH14 causative variants may be more contributory and emphasize the importance of considering this gene in patients with nonsyndromic mainly post-lingual severe form of hearing loss. However, further functional studies are needed to confirm the pathogenicity of these variants.

S100A11 promotes focal adhesion disassembly via myosin II-driven contractility and Piezo1-mediated Ca2+ entry.

S100A11 is a small Ca2+-activatable protein known to localize along stress fibers (SFs). Analyzing S100A11 localization in HeLa and U2OS cells further revealed S100A11 enrichment at focal adhesions (FAs). Strikingly, S100A11 levels at FAs increased sharply, yet transiently, just before FA disassembly. Elevating intracellular Ca2+ levels with ionomycin stimulated both S100A11 recruitment and subsequent FA disassembly. However, pre-incubation with the non-muscle myosin II (NMII) inhibitor blebbistatin or with an inhibitor of the stretch-activatable Ca2+ channel Piezo1 suppressed S100A11 recruitment, implicating S100A11 in an actomyosin-driven FA recruitment mechanism involving Piezo1-dependent Ca2+ influx. Applying external forces on peripheral FAs likewise recruited S100A11 to FAs even if NMII activity was inhibited, corroborating the mechanosensitive recruitment mechanism of S100A11. However, extracellular Ca2+ and Piezo1 function were indispensable, indicating that NMII contraction forces act upstream of Piezo1-mediated Ca2+ influx, in turn leading to S100A11 activation and FA recruitment. S100A11-knockout cells display enlarged FAs and had delayed FA disassembly during cell membrane retraction, consistent with impaired FA turnover in these cells. Our results thus demonstrate a novel function for S100A11 in promoting actomyosin contractility-driven FA disassembly.

Role of ZIP kinase in development of myofibroblast differentiation from HPMCs.

During the development of pleural fibrosis, pleural mesothelial cells (PMCs) undergo phenotypic switching from differentiated mesothelial cells to mesenchymal cells (MesoMT). Here, we investigated how external stimuli such as TGF-ß induce HPMC-derived myofibroblast differentiation to facilitate the development of pleural fibrosis. TGF-ß significantly increased di-phosphorylation but not mono-phosphorylation of myosin II regulatory light chain (RLC) in HPMCs. An increase in RLC di-phosphorylation was also found at the pleural layer of our carbon black bleomycin (CBB) pleural fibrosis mouse model, where it showed filamentous localization that coincided with alpha smooth muscle actin (αSMA) in the cells in the pleura. Among the protein kinases that can phosphorylate myosin II RLC, ZIPK (zipper-interacting kinase) protein expression was significantly augmented after TGF-ß stimulation. Furthermore, ZIPK gene silencing attenuated RLC di-phosphorylation, suggesting that ZIPK is responsible for di-phosphorylation of myosin II in HPMCs. Although TGF-ß significantly increased the expression of ZIP kinase protein, the change in ZIP kinase mRNA was marginal, suggesting a posttranscriptional mechanism for the regulation of ZIP kinase expression by TGF-ß. ZIPK gene knockdown (KD) also significantly reduced TGF-ß-induced upregulation of αSMA expression. This finding suggests that siZIPK attenuates myofibroblast differentiation of HPMCs. siZIPK diminished TGF-ß-induced contractility of HPMCs consistent with siZIPK-induced decrease in the di-phosphorylation of myosin II RLC. The present results implicate ZIPK in the regulation of the contractility of HPMC-derived myofibroblasts, phenotype switching, and myofibroblast differentiation of HPMCs.NEW & NOTEWORTHY Here, we highlight that ZIP kinase is responsible for di-phosphorylation of myosin light chain, which facilitates stress fiber formation and actomyosin-based cell contraction during mesothelial to mesenchymal transition in human pleural mesothelial cells. This transition has a significant impact on tissue remodeling and subsequent stiffness of the pleura. This study provides insight into a new therapeutic strategy for the treatment of pleural fibrosis.