There are three main classes of actin nucleation factors: Arp2/3 complexes, Spire and Formin. Spire assembles microfilaments by nucleating stable longitudinal tetramers and binding actin to the growing end of the microfilament. As early as 1999, Wellington et al. identified Spire as an actin nucleating agent, however, over the years, most studies have focused on Arp2/3 and Formin proteins; there has been relatively less research on Spire as a member of the actin nucleating factors. Recent studies have shown that Spire is involved in the vesicular transport through the synthesis of actin and plays an important role in neural development. In this paper, we reviewed the structure, expression and function of Spire, and its association with disease in order to identify meaningful potential directions for studies on Spire.

Mammals have 6 highly conserved actin isoforms with nonredundant biological functions. The molecular basis of isoform specificity, however, remains elusive due to a lack of tools. Here, we describe the development of IntAct, an internal tagging strategy to study actin isoforms in fixed and living cells. We identified a residue pair in β-actin that permits tag integration and used knock-in cell lines to demonstrate that IntAct β-actin expression and filament incorporation is indistinguishable from wild type. Furthermore, IntAct β-actin remains associated with common actin-binding proteins (ABPs) and can be targeted in living cells. We demonstrate the usability of IntAct for actin isoform investigations by showing that actin isoform-specific distribution is maintained in human cells. Lastly, we observed a variant-dependent incorporation of tagged actin variants into yeast actin patches, cables, and cytokinetic rings demonstrating cross species applicability. Together, our data indicate that IntAct is a versatile tool to study actin isoform localization, dynamics, and molecular interactions.

The inner lining of blood vessels, the endothelium, is made up of endothelial cells. Vascular endothelial (VE)-cadherin protein forms a bond with VE-cadherin from neighboring cells to determine the size of gaps between the cells and thereby regulate the size of particles that can cross the endothelium. Chemical cues such as thrombin, along with mechanical properties of the cell and extracellular matrix are known to affect the permeability of endothelial cells. Abnormal permeability is found in patients suffering from diseases including cardiovascular diseases, cancer, and COVID-19. Even though some of the regulatory mechanisms affecting endothelial permeability are well studied, details of how several mechanical and chemical stimuli acting simultaneously affect endothelial permeability are not yet understood. In this article, we present a continuum-level mechanical modeling framework to study the highly dynamic nature of the VE-cadherin bonds. Taking inspiration from the catch-slip behavior that VE-cadherin complexes are known to exhibit, we model the VE-cadherin homophilic bond as cohesive contact with damage following a traction-separation law. We explicitly model the actin cytoskeleton and substrate to study their role in permeability. Our studies show that mechanochemical coupling is necessary to simulate the influence of the mechanical properties of the substrate on permeability. Simulations show that shear between cells is responsible for the variation in permeability between bicellular and tricellular junctions, explaining the phenotypic differences observed in experiments. An increase in the magnitude of traction force due to disturbed flow that endothelial cells experience results in increased permeability, and it is found that the effect is higher on stiffer extracellular matrix. Finally, we show that the cylindrical monolayer exhibits higher permeability than the planar monolayer under unconstrained cases. Thus, we present a contact mechanics-based mechanochemical model to investigate the variation in the permeability of endothelial monolayer due to multiple loads acting simultaneously.

Hypertrophic cardiomyopathy (HCM) is a common inherited cardiac disorder characterized by marked clinical and genetic heterogeneity. Ethnic groups underrepresented in studies may have distinctive characteristics. We sought to evaluate the clinical and genetic landscape of Russian HCM patients. A total of 193 patients (52% male; 95% Eastern Slavic origin; median age 56 years) were clinically evaluated, including genetic testing, and prospectively followed to document outcomes. As a result, 48% had obstructive HCM, 25% had HCM in family, 21% were asymptomatic, and 68% had comorbidities. During 2.8 years of follow-up, the all-cause mortality rate was 2.86%/year. A total of 5.7% received an implantable cardioverter-defibrillator (ICD), and 21% had septal reduction therapy. A sequencing analysis of 176 probands identified 64 causative variants in 66 patients (38%); recurrent variants were MYBPC3 p.Q1233* (8), MYBPC3 p.R346H (2), MYH7 p.A729P (2), TPM1 p.Q210R (3), and FLNC p.H1834Y (2); 10 were multiple variant carriers (5.7%); 5 had non-sarcomeric HCM, ALPK3, TRIM63, and FLNC. Thin filament variant carriers had a worse prognosis for heart failure (HR = 7.9, p = 0.007). In conclusion, in the Russian HCM population, the low use of ICD and relatively high mortality should be noted by clinicians; some distinct recurrent variants are suspected to have a founder effect; and family studies on some rare variants enriched worldwide knowledge in HCM.

Cells rely on their cytoskeleton for key processes including division and directed motility. Actin filaments are a primary constituent of the cytoskeleton. Although actin filaments can create a variety of network architectures linked to distinct cell functions, the microscale molecular interactions that give rise to these macroscale structures are not well understood. In this work, we investigate the microscale mechanisms that produce different branched actin network structures using an iterative classification approach. First, we employ a simple yet comprehensive agent-based model that produces synthetic actin networks with precise control over the microscale dynamics. Then we apply machine learning techniques to classify actin networks based on measurable network density and geometry, identifying key mechanistic processes that lead to particular branched actin network architectures. Extensive computational experiments reveal that the most accurate method uses a combination of supervised learning based on network density and unsupervised learning based on network symmetry. This framework can potentially serve as a powerful tool to discover the molecular interactions that produce the wide variety of actin network configurations associated with normal development as well as pathological conditions such as cancer.

The satisfactory visualization of cytoskeletal components in the brain is challenging. The ubiquitous distribution of the networks of microtubules, microfilaments, and intermediate filaments in all the neural tissues, together with the variability in the outcomes of fluorescent protein fusion strategies and their limited applicability to dynamic studies of antibodies and drugs as chromophore vehicles, make classical optical approaches not as effective as for other proteins. When tubulin needs to be studied, the label-free generation of second harmonics is a very suitable option due to the non-centrosymmetric organization of the molecule. This technique, when conjugated to microscopy, can qualitatively describe the volumetric distribution of parallel bundles of microtubules in biological samples, with the additional advantage of working with fresh tissues that are unfixed and unpermeabilized. This work describes how to image tubulin with a commercial second harmonic generation microscopy setup to highlight microtubules in the tubulin-enriched structures of the oligodendrocytes, as in hypomyelination with atrophy of the basal ganglia and cerebellum (H-ABC) tubulinopathy, a recently described myelin disorder.

Signaling molecules are crucial to perceive and translate intra- and extracellular cues. Phosphoinositides and the proteins responsible for their biosynthesis (e.g., lipid kinases) are known to influence the (re)organization of cytoskeletal elements, namely, through interaction with actin and actin-binding proteins. Here we describe methods to functionally characterize lipid kinases and their phosphoinositide metabolites in relation to actin dynamics. These methods include GFP-tagged protein expression followed by time-resolved live imaging and quantitative image analysis. When combined with biochemical and interaction studies, these methods can be used to correlate signaling with actin dynamics, microfilament assembly, and intracellular trafficking, linking structure and function.

Progress in cytoskeletal research in animal systems has been accompanied by the development of single-cell systems (e.g., fibroblasts in culture). Single-cell systems exist for plant research, but the presence of a cell wall hinders the possibility to relate cytoskeleton dynamics to changes in cell shape or in mechanical stress pattern. Here we present two protocols to confine wall-less plant protoplasts in microwells with defined geometries. Either protocol might be more or less adapted to the question at hand. For instance, when using microwells made of agarose, the composition of the well can be easily modified to analyze the impact of biochemical cues. When using microwells in a stiff polymer (NOA73), protoplasts can be pressurized, and the wall of the well can be coated with cell wall components. Using both protocols, we could analyze microtubule and actin dynamics in vivo while also revealing the relative contribution of geometry and stress in their self-organization.

Fascin is a filamentous actin (F-actin) bundling protein, which cross-links F-actin into bundles and becomes an important component of filopodia on the cell surface. Fascin is overexpressed in many types of cancers. The mutation of fascin affects its ability to bind to F-actin and the progress of cancer. In this paper, we have studied the effects of residues of K22, K41, K43, K241, K358, K399, and K471 using molecular dynamics (MD) simulation. For the strong-effect residues, that is, K22, K41, K43, K358, and K471, our results show that the mutation of K to A leads to large values of root mean square fluctuation (RMSF) around the mutated residues, indicating those residues are important for the flexibility and thermal stability. On the other hand, based on residue cross-correlation analysis, alanine mutations of these residues reinforce the correlation between residues. Together with the RMSF data, the local flexibility is extended to the entire protein by the strong correlations to influence the dynamics and function of fascin. By contrast, for the mutants of K241A and K399A those do not affect the function of fascin, the RMSF data do not show significant differences compared with wild-type fascin. These findings are in a good agreement with experimental studies.

F-actin dynamics (polymerization and depolymerization) are associated with nucleotide exchange, providing the driving forces for dynamic cellular activities. As an important residue in the nucleotide state-sensing region in actin, His73 is often found to be methylated in natural actin and directly participates in F-actin dynamics by regulating nucleotide exchange. The interaction between His73 and its neighboring residue, Gly158, has significance for F-actin dynamics. However, this weak chemical interaction is difficult to characterize using classic molecular modeling methods. In this study, ab initio modeling was employed to explore the binding energy between His73 and Gly158. The results confirm that the methyl group on the His73 side chain contributes to the structural stability of atomistic networks in the nucleotide state-sensing region of actin monomers and confines the material exchange (Pi release) pathway within F-actin dynamics. Further binding energy analyses of actin structures under different nucleotide states showed that the potential model of His73/Gly158 hydrogen bond breaking in the material exchange mechanism is not obligatory within F-actin dynamics.