Chloride intracellular channel (CLIC) proteins function as fusogens.

Chloride Intracellular Channel (CLIC) family members uniquely transition between soluble and membrane-associated conformations. Despite decades of extensive functional and structural studies, CLICs' function as ion channels remains debated, rendering our understanding of their physiological role incomplete. Here, we expose the function of CLIC5 as a fusogen. We demonstrate that purified CLIC5 directly interacts with the membrane and induces fusion, as reflected by increased liposomal diameter and lipid and content mixing between liposomes. Moreover, we show that this activity is facilitated by acidic pH, a known trigger for CLICs' transition to a membrane-associated conformation, and that increased exposure of the hydrophobic inter-domain interface is crucial for this process. Finally, mutation of a conserved hydrophobic interfacial residue diminishes the fusogenic activity of CLIC5 in vitro and impairs excretory canal extension in C. elegans in vivo. Together, our results unravel the long-sought physiological role of these enigmatic proteins.

Therapeutic gene silencing of CKAP5 leads to lethality in genetically unstable cancer cells.

The potential of microtubule-associated protein targets for cancer therapeutics remains largely unexplored due to the lack of target-specific agents. Here, we explored the therapeutic potential of targeting cytoskeleton-associated protein 5 (CKAP5), an important microtubule-associated protein, with CKAP5-targeting siRNAs encapsulated in lipid nanoparticles (LNPs). Our screening of 20 solid cancer cell lines demonstrated selective vulnerability of genetically unstable cancer cell lines in response to CKAP5 silencing. We identified a highly responsive chemo-resistant ovarian cancer cell line, in which CKAP5 silencing led to significant loss in EB1 dynamics during mitosis. Last, we demonstrated the therapeutic potential in an in vivo ovarian cancer model, showing 80% survival rate of siCKAP5 LNPs-treated animals. Together, our results highlight the importance of CKAP5 as a therapeutic target for genetically unstable ovarian cancer and warrants further investigation into its mechanistic aspects.

Actin-capping protein regulates actomyosin contractility to maintain germline architecture in C. elegans.

Actin dynamics play an important role in tissue morphogenesis, yet the control of actin filament growth takes place at the molecular level. A challenge in the field is to link the molecular function of actin regulators with their physiological function. Here, we report an in vivo role of the actin-capping protein CAP-1 in the Caenorhabditis elegans germline. We show that CAP-1 is associated with actomyosin structures in the cortex and rachis, and its depletion or overexpression led to severe structural defects in the syncytial germline and oocytes. A 60% reduction in the level of CAP-1 caused a twofold increase in F-actin and non-muscle myosin II activity, and laser incision experiments revealed an increase in rachis contractility. Cytosim simulations pointed to increased myosin as the main driver of increased contractility following loss of actin-capping protein. Double depletion of CAP-1 and myosin or Rho kinase demonstrated that the rachis architecture defects associated with CAP-1 depletion require contractility of the rachis actomyosin corset. Thus, we uncovered a physiological role for actin-capping protein in regulating actomyosin contractility to maintain reproductive tissue architecture.

Tension-dependent RHGF-1 recruitment to stress fibers drives robust spermathecal tissue contraction.

Contractile epithelial tubes are found in various organs, such as lung airways and blood capillaries. Their ability to sense luminal pressure and respond with adequate contractility is essential for their physiology, and its mis-regulation results in diseases such as asthma and hypertension. Here, we describe a mechanoresponsive regulatory pathway downstream of tissue stretching that controls contraction of the C. elegans spermatheca, a tubular structure where fertilization occurs. Using live-imaging, we show that ovulation-induced stretching of spermathecal cells leads to recruitment of the RhoGEF RHGF-1 to stress fibers, which activates RHO-1 and myosin II in a positive feedback loop. Through deletion analysis, we identified the PDZ domain of RHGF-1 as responsible for F-actin binding, and genetic epistasis analysis with the RhoGAP spv-1 demonstrated that tension-dependent recruitment of RHGF-1 to F-actin is required for robust spermathecal contractility. Our study illustrates how mechanosensitive regulators of Rho GTPases provide epithelial tubes the ability to tune their contractility in response to internal pressure.

Directed cell invasion and asymmetric adhesion drive tissue elongation and turning in C. elegans gonad morphogenesis.

Development of the C. elegans gonad has long been studied as a model of organogenesis driven by collective cell migration. A somatic cell named the distal tip cell (DTC) is thought to serve as the leader of following germ cells; yet, the mechanism for DTC propulsion and maneuvering remains elusive. Here, we demonstrate that the DTC is not self-propelled but rather is pushed by the proliferating germ cells. Proliferative pressure pushes the DTC forward, against the resistance of the basement membrane in front. The DTC locally secretes metalloproteases that degrade the impeding membrane, resulting in gonad elongation. Turning of the gonad is achieved by polarized DTC-matrix adhesions. The asymmetrical traction results in a bending moment on the DTC. Src and Cdc42 regulate integrin adhesion polarity, whereas an external netrin signal determines DTC orientation. Our findings challenge the current view of DTC migration and offer a distinct framework to understand organogenesis.

Visualizing and quantifying molecular and cellular processes in Caenorhabditis elegans using light microscopy.

Light microscopes are the cell and developmental biologists' "best friend," providing a means to see structures and follow dynamics from the protein to the organism level. A huge advantage of Caenorhabditis elegans as a model organism is its transparency, which coupled with its small size means that nearly every biological process can be observed and measured with the appropriate probe and light microscope. Continuous improvement in microscope technologies along with novel genome editing techniques to create transgenic probes have facilitated the development and implementation of a dizzying array of methods for imaging worm embryos, larvae, and adults. In this review, we provide an overview of the molecular and cellular processes that can be visualized in living worms using light microscopy. A partial inventory of fluorescent probes and techniques successfully used in worms to image the dynamics of cells, organelles, DNA, and protein localization and activity is followed by a practical guide to choosing between various imaging modalities, including widefield, confocal, lightsheet, and structured illumination microscopy. Finally, we discuss the available tools and approaches, including machine learning, for quantitative image analysis tasks, such as colocalization, segmentation, object tracking, and lineage tracing. Hopefully, this review will inspire worm researchers who have not yet imaged their worms to begin, and push those who are imaging to go faster, finer, and longer.

Levodopa-responsive dystonia caused by biallelic PRKN exon inversion invisible to exome sequencing.

Biallelic pathogenic variants in PRKN (PARK2), encoding the E3 ubiquitin ligase parkin, lead to early-onset Parkinson's disease. Structural variants, including duplications or deletions, are common in PRKN due to their location within the fragile site FRA6E. These variants are readily detectable by copy number variation analysis. We studied four siblings with levodopa-responsive dystonia by exome sequencing followed by genome sequencing. Affected individuals developed juvenile levodopa-responsive dystonia with subsequent appearance of parkinsonism and motor fluctuations that improved by subthalamic stimulation. Exome sequencing and copy number variation analysis were not diagnostic, yet revealed a shared homozygous block including PRKN. Genome sequencing revealed an inversion within PRKN, with intronic breakpoints flanking exon 5. Breakpoint junction analysis implicated non-homologous end joining and possibly replicative mechanisms as the repair pathways involved. Analysis of cDNA indicated skipping of exon 5 (84 bp) that was replaced by 93 bp of retained intronic sequence, preserving the reading frame yet altering a significant number of residues. Balanced copy number inversions in PRKN are associated with a severe phenotype. Such structural variants, undetected by exome analysis and by copy number variation analysis, should be considered in the relevant clinical setting. These findings raise the possibility that PRKN structural variants are more common than currently estimated.

Probing the effect of clustering on EphA2 receptor signaling efficiency by subcellular control of ligand-receptor mobility.

Clustering of ligand:receptor complexes on the cell membrane is widely presumed to have functional consequences for subsequent signal transduction. However, it is experimentally challenging to selectively manipulate receptor clustering without altering other biochemical aspects of the cellular system. Here, we develop a microfabrication strategy to produce substrates displaying mobile and immobile ligands that are separated by roughly 1 µm, and thus experience an identical cytoplasmic signaling state, enabling precision comparison of downstream signaling reactions. Applying this approach to characterize the ephrinA1:EphA2 signaling system reveals that EphA2 clustering enhances both receptor phosphorylation and downstream signaling activity. Single-molecule imaging clearly resolves increased molecular binding dwell times at EphA2 clusters for both Grb2:SOS and NCK:N-WASP signaling modules. This type of intracellular comparison enables a substantially higher degree of quantitative analysis than is possible when comparisons must be made between different cells and essentially eliminates the effects of cellular response to ligand manipulation.

Pyk2 regulates cell-edge protrusion dynamics by interacting with Crk.

Focal adhesion kinase (FAK) is well established as a regulator of cell migration, but whether and how the closely related proline-rich tyrosine kinase 2 (Pyk2) regulates fibroblast motility is still under debate. Using mouse embryonic fibroblasts (MEFs) from Pyk2-/- mice, we show here, for the first time, that lack of Pyk2 significantly impairs both random and directed fibroblast motility. Pyk2-/- MEFs show reduced cell-edge protrusion dynamics, which is dependent on both the kinase and protein-protein binding activities of Pyk2. Using bioinformatics analysis of in vitro high- throughput screens followed by text mining, we identified CrkI/II as novel substrates and interactors of Pyk2. Knockdown of CrkI/II shows altered dynamics of cell-edge protrusions, which is similar to the phenotype observed in Pyk2-/- MEFs. Moreover, epistasis experiments suggest that Pyk2 regulates the dynamics of cell-edge protrusions via direct and indirect interactions with Crk that enable both activation and down-regulation of Crk-mediated cytoskeletal signaling. This complex mechanism may enable fine-tuning of cell-edge protrusion dynamics and consequent cell migration on the one hand together with tight regulation of cell motility, a process that should be strictly limited to specific time and context in normal cells, on the other hand.

Mechanosensing in embryogenesis.

Mechanical forces generated by living cells at the molecular level propagate to the cellular and organismal level and have profound consequences for embryogenesis. A direct result of force application is movement, as occurs in chromosome separation, cell migration, or tissue folding. A less direct, but equally important effect of force, is the activation of mechanosensitive signaling, which allows cells to probe their mechanical surrounding and communicate with each other over short and long distances. In this review, we focus on forces as a means of conveying information and affecting cell behavior during embryogenesis. We discuss four developmental processes that demonstrate the involvement of force in cell fate determination, growth, morphogenesis, and organogenesis, in a variety of model organisms. Finally, a generalizable pathway of mechanosensing and mechanotransduction in vivo is described, and we highlight similarities between morphogens and forces in patterning of embryos.

Thymosin ß4 is essential for adherens junction stability and epidermal planar cell polarity.

Planar cell polarity (PCP) is essential for tissue morphogenesis and homeostasis; however, the mechanisms that orchestrate the cell shape and packing dynamics required to establish PCP are poorly understood. Here, we identified a major role for the globular (G)-actin-binding protein thymosin-ß4 (TMSB4X) in PCP establishment and cell adhesion in the developing epidermis. Depletion of Tmsb4x in mouse embryos hindered eyelid closure and hair-follicle angling owing to PCP defects. Tmsb4x depletion did not preclude epidermal cell adhesion in vivo or in vitro; however, it resulted in abnormal structural organization and stability of adherens junction (AJ) due to defects in filamentous (F)-actin and G-actin distribution. In cultured keratinocytes, TMSB4X depletion increased the perijunctional G/F-actin ratio and decreased G-actin incorporation into junctional actin networks, but it did not change the overall actin expression level or cellular F-actin content. A pharmacological treatment that increased the G/F-actin ratio and decreased actin polymerization mimicked the effects of Tmsb4x depletion on both AJs and PCP. Our results provide insights into the regulation of the actin pool and its involvement in AJ function and PCP establishment.

Balloon Eustachian tuboplasty in a professional Navy SEAL diver: case report.

Middle ear barotrauma due to dilatory Eustachian tube dysfunction (ETD) is probably the most common medical disorder related to diving. Moreover, ETD makes divers prone to other diving-related accidents, including inner ear barotrauma and alternobaric vertigo. Until the development of Eustachian tube balloon dilation no diving-compatible surgical options existed to effectively and safely prevent recurrence. We present a case of an Israeli Navy SEAL diver who dives in extreme strenuous combat-related closed-circuit rebreather (CCR) dives. Due to repeated middle ear barotrauma, the patient underwent Eustachian tube balloon dilation of the affected side. Following surgery, the patient returned to both CCR and scuba dives but still suffered from middle ear symptoms and repeated barotrauma hence was eventually disqualified from further combat diving.

Diverse roles of non-muscle myosin II contractility in 3D cell migration.

All is flux, nothing stays still. Heraclitus of Ephesus' characterization of the universe holds true for cells within animals and for proteins within cells. In this review, we examine the dynamics of actin and non-muscle myosin II within cells, and how their dynamics power the movement of cells within tissues. The 3D environment that migrating cells encounter along their path also changes over time, and cells can adopt various mechanisms of motility, depending on the topography, mechanics and chemical composition of their surroundings. We describe the differential spatio-temporal regulation of actin and myosin II-mediated contractility in mesenchymal, lobopodial, amoeboid, and swimming modes of cell migration. After briefly reviewing the biochemistry of myosin II, we discuss the role actomyosin contractility plays in the switch between modes of 3D migration that cells use to adapt to changing environments.

Germ Granules Govern Small RNA Inheritance.

In C. elegans nematodes, components of liquid-like germ granules were shown to be required for transgenerational small RNA inheritance. Surprisingly, we show here that mutants with defective germ granules can nevertheless inherit potent small RNA-based silencing responses, but some of the mutants lose this ability after many generations of homozygosity. Animals mutated in pptr-1, which is required for stabilization of P granules in the early embryo, display extraordinarily strong heritable RNAi responses, lasting for tens of generations. Intriguingly, the RNAi capacity of descendants derived from mutants defective in the core germ granule proteins MEG-3 and MEG-4 is determined by the genotype of the ancestors and changes transgenerationally. Further, whether the meg-3/4 mutant alleles were present in the paternal or maternal lineages leads to different transgenerational consequences. Small RNA inheritance, rather than maternal contribution of the germ granules themselves, mediates the transgenerational defects in RNAi of meg-3/4 mutants and their progeny. Accordingly, germ granule defects lead to heritable genome-wide mis-expression of endogenous small RNAs. Upon disruption of germ granules, hrde-1 mutants can inherit RNAi, although HRDE-1 was previously thought to be absolutely required for RNAi inheritance. We propose that germ granules sort and shape the RNA pool, and that small RNA inheritance maintains this activity for multiple generations.

Reciprocal regulation of actomyosin organization and contractility in nonmuscle cells by tropomyosins and alpha-actinins.

Contractile arrays of actin and myosin II filaments drive many essential processes in nonmuscle cells, including migration and adhesion. Sequential organization of actin and myosin along one dimension is followed by expansion into a two-dimensional network of parallel actomyosin fibers, in which myosin filaments are aligned to form stacks. The process of stack formation has been studied in detail. However, factors that oppose myosin stack formation have not yet been described. Here, we show that tropomyosins act as negative regulators of myosin stack formation. Knockdown of any or all tropomyosin isoforms in rat embryonic fibroblasts resulted in longer and more numerous myosin stacks and a highly ordered actomyosin organization. The molecular basis for this, we found, is the competition between tropomyosin and alpha-actinin for binding actin. Surprisingly, excessive order in the actomyosin network resulted in smaller focal adhesions, lower tension within the network, and smaller traction forces. Conversely, disordered actomyosin bundles induced by alpha-actinin knockdown led to higher than normal tension and traction forces. Thus, tropomyosin acts as a check on alpha-actinin to achieve intermediate levels of myosin stacks matching the force requirements of the cell.

Salmonella biofilms program innate immunity for persistence in Caenorhabditis elegans.

The adaptive in vivo mechanisms underlying the switch in Salmonella enterica lifestyles from the infectious form to a dormant form remain unknown. We employed Caenorhabditis elegans as a heterologous host to understand the temporal dynamics of Salmonella pathogenesis and to identify its lifestyle form in vivo. We discovered that Salmonella exists as sessile aggregates, or in vivo biofilms, in the persistently infected C. elegans gut. In the absence of in vivo biofilms, Salmonella killed the host more rapidly by actively inhibiting innate immune pathways. Regulatory cross-talk between two major Salmonella pathogenicity islands, SPI-1 and SPI-2, was responsible for biofilm-induced changes in host physiology during persistent infection. Thus, biofilm formation is a survival strategy in long-term infections, as prolonging host survival is beneficial for the parasitic lifestyle.

From cell shape to cell fate via the cytoskeleton - Insights from the epidermis.

Animal cells exhibit a wide range of shapes that reflect their diverse functions. Cell shape is determined by a balance between internal and external forces and therefore involves the cytoskeleton and its associated adhesion structures. Cell shape dynamics during development and homeostasis are tightly regulated and closely coordinated with cell fate determination. Defects in cell shape are a hallmark of many pathological conditions including cancer and skin diseases. This review highlights the links between cell shape and cell fate in the epidermis, which have been studied for over 40 years both in vitro and in vivo. Briefly discussing seminal experiments showing the strong coupling between keratinocyte cell shape and their fate we primarily focus on recent studies uncovering novel cellular and molecular mechanisms linking epidermal cell shape with cell growth, differentiation, asymmetric division, and delamination.

The RhoGAP SPV-1 regulates calcium signaling to control the contractility of the Caenorhabditis elegans spermatheca during embryo transits.

Contractility of the nonmuscle and smooth muscle cells that comprise biological tubing is regulated by the Rho-ROCK (Rho-associated protein kinase) and calcium signaling pathways. Although many molecular details about these signaling pathways are known, less is known about how they are coordinated spatiotemporally in biological tubes. The spermatheca of the Caenorhabditis elegans reproductive system enables study of the signaling pathways regulating actomyosin contractility in live adult animals. The RhoGAP (GTPase--activating protein toward Rho family small GTPases) SPV-1 was previously identified as a negative regulator of RHO-1/Rho and spermathecal contractility. Here, we uncover a role for SPV-1 as a key regulator of calcium signaling. spv-1 mutants expressing the calcium indicator GCaMP in the spermatheca exhibit premature calcium release, elevated calcium levels, and disrupted spatial regulation of calcium signaling during spermathecal contraction. Although RHO-1 is required for spermathecal contractility, RHO-1 does not play a significant role in regulating calcium. In contrast, activation of CDC-42 recapitulates many aspects of spv-1 mutant calcium signaling. Depletion of cdc-42 by RNA interference does not suppress the premature or elevated calcium signal seen in spv-1 mutants, suggesting other targets remain to be identified. Our results suggest that SPV-1 works through both the Rho-ROCK and calcium signaling pathways to coordinate cellular contractility.

Principles of Actomyosin Regulation In Vivo.

The actomyosin cytoskeleton is responsible for most force-driven processes in cells and tissues. How it assembles into the necessary structures at the right time and place is an important question. Here, we focus on molecular mechanisms of actomyosin regulation recently elucidated in animal models, and highlight several common principles that emerge. The architecture of the actomyosin network - an important determinant of its function - results from actin polymerization, crosslinking and turnover, localized myosin activation, and contractility-driven self-organization. Spatiotemporal regulation is achieved by tissue-specific expression and subcellular localization of Rho GTPase regulators. Subcellular anchor points of actomyosin structures control the outcome of their contraction, and molecular feedback mechanisms dictate whether they are transient, cyclic, or persistent.

Syncytial germline architecture is actively maintained by contraction of an internal actomyosin corset.

Syncytial architecture is an evolutionarily-conserved feature of the germline of many species and plays a crucial role in their fertility. However, the mechanism supporting syncytial organization is largely unknown. Here, we identify a corset-like actomyosin structure within the syncytial germline of Caenorhabditis elegans, surrounding the common rachis. Using laser microsurgery, we demonstrate that actomyosin contractility within this structure generates tension both in the plane of the rachis surface and perpendicular to it, opposing membrane tension. Genetic and pharmacological perturbations, as well as mathematical modeling, reveal a balance of forces within the gonad and show how changing the tension within the actomyosin corset impinges on syncytial germline structure, leading, in extreme cases, to sterility. Thus, our work highlights a unique tissue-level cytoskeletal structure, and explains the critical role of actomyosin contractility in the preservation of a functional germline.