Functional interdependence of the actin regulators CAP1 and cofilin1 in control of dendritic spine morphology

The vast majority of excitatory synapses are formed on small dendritic protrusions termed dendritic spines. Dendritic spines vary in size and density that are both crucial determinants of excitatory synaptic transmission. Aberrations in spine morphogenesis can compromise brain function and have been associated with neuropsychiatric disorders. Because actin filaments (F-actin) are the major structural component in spines, actin-binding proteins (ABP) that control F-actin dis-/assembly moved into the focus as critical regulators of brain function. Indeed, mouse studies identified the ABP cofilin1 as a key regulator of spine morphology, synaptic transmission and behavior. These studies emphasized the necessity for a tight control of cofilin1 to ensure proper brain function. We report spine enrichment of cyclase-associated protein 1 (CAP1), a conserved multidomain protein with largely unknown physiological functions. Super-resolution microscopy and live cell imaging of CAP1-deficient hippocampal neurons revealed impaired synaptic F-actin organization and dynamics associated with alterations in spine morphology. Mechanistically, we found that CAP1 cooperated with cofilin1 in spines and that its helical folded domain mediated this interaction. Moreover, our data proved functional interdependence of CAP1 and cofilin1 in control of spine morphology. In summary, we identified CAP1 as a novel regulator of the postsynaptic actin cytoskeleton that was essential for synaptic cofilin1 activity.

PA1 Participates in the Maintenance of Blood-Testis Barrier Integrity via Cooperation with JUN in the Sertoli Cells of Mice.

BackgroundThe blood-testis barrier (BTB) is essential to the microenvironment of spermatogenesis, and Sertoli cells provide the cellular basis for BTB construction. Numerous nuclear transcription factors have been identified to be vital for the proper functioning of Sertoli cells. PA1 has been reported to play important roles during diverse biological processes, yet its potential function in male reproduction is still unknown. ResultsHere, we show that PA1 was highly expressed in human and mouse testis and predominantly localized in the nuclei of Sertoli cells. Sertoli cell-specific Pa1 knockout resulted in an azoospermia-like phenotype in mice. The knockout of this gene led to multiple defects in spermatogenesis, such as the disorganization of the cytoskeleton during basal and apical ectoplasmic specialization and the disruption of the BTB. Further transcriptomic analysis, together with Cut-Tag results of PA1 in Sertoli cells, revealed that PA1 could affect the expression of a subset of genes that are essential for the normal function of Sertoli cells, including those genes associated with actin organization and cellular junctions such as Connexin43 (Cx43). We further demonstrated that the expression of Cx43 depended on the interaction between JUN, one of the AP-1 complex transcription factors, and PA1. ConclusionOverall, our findings reveal that PA1 is essential for the maintenance of BTB integrity in Sertoli cells and regulates BTB construction-related gene expression via transcription factors. Thus, this newly discovered mechanism in Sertoli cells provides a potential diagnostic or even therapeutic target for some individuals with azoospermia.

Multiparameters dependance of tissue shape maintenance in myoblasts multicellular aggregates: the role of intermediate filaments.

Liquid and elastic behavior of tissues drives their morphology and their response to the environment. They appear as the first insight on tissue mechanics. We explore the role of individual cell properties on spheroids of mouse muscle precursor cells by developing a fully automated surface tension and Young's modulus measurement system. Flattening multicellular aggregates under magnetic constraint, we show that rigidity and surface tension act as highly sensitive macroscopic reporters closely related to microscopic local tension and effective adhesion. Shedding light on the major contributions of acto-myosin contractility, actin organization and intercellular adhesions, we reveal the role of desmin organization on the macroscopic mechanics of this tissue model.

Role for the phosphatidylinositol 3-phosphate 5-kinase in antifungal tolerance in Candida glabrata

Candida glabrata is an opportunistic fungal pathogen of humans, which is intrinsically less susceptible to widely used azole antifungals, that block ergosterol biosynthesis. The major azole resistance mechanisms include mitochondrial dysfunction and multidrug efflux pump overexpression. In the current study, we have uncovered an essential role for the actin cytoskeletal network reorganization in survival of the azole stress. We demonstrate for the first time that the azole antifungal fluconazole induces remodelling of the actin cytoskeleton in C. glabrata, and genetic or chemical perturbation of actin structures results in intracellular sterol accumulation and azole susceptibility. Further, we showed that the vacuolar membrane-resident phosphatidylinositol 3-phosphate 5-kinase (CgFab1) is pivotal to this process, as CgFAB1 disruption impaired vacuole homeostasis and actin organization. We also showed that the actin depolymerization factor CgCof1 binds to phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2), and CgCof1 distribution along with the actin filament-capping protein CgCap2 is altered upon both CgFAB1disruption and fluconazole exposure. Additionally, while the F-actin-stabilizing compound jasplakinolide rescued azole toxicity in cytoskeleton defective-mutants, the actin polymerization inhibitor latrunculin B rendered fluconazole fully and partially fungicidal in azole-susceptible and azole-resistant C. glabrata clinical isolates, respectively. These data underscore the essentiality of actin cytoskeleton reorganization for azole stress survival. Lastly, we have also shown a pivotal role of CgFab1 kinase activity regulators, CgFig4, CgVac7 and CgVac14, through genetic analysis, in azole and echinocandin antifungal tolerance. Altogether, I shall present our findings on functions and metabolism of the PI(3,5)P2 lipid in antifungal tolerance and virulence of C. glabrata.

Bundling up the Role of the Actin Cytoskeleton in Primary Root Growth

Primary root growth is required by the plant to anchor in the soil and reach out for nutrients and water, while dealing with obstacles. Efficient root elongation and bending depends upon the coordinated action of environmental sensing, signal transduction, and growth responses. The actin cytoskeleton is a highly plastic network that constitutes a point of integration for environmental stimuli and hormonal pathways. In this review, we present a detailed compilation highlighting the importance of the actin cytoskeleton during primary root growth and we describe how actin-binding proteins, plant hormones, and actin-disrupting drugs affect root growth and root actin. We also discuss the feedback loop between actin and root responses to light and gravity. Actin affects cell division and elongation through the control of its own organization. We remark upon the importance of longitudinally oriented actin bundles as a hallmark of cell elongation as well as the role of the actin cytoskeleton in protein trafficking and vacuolar reshaping during this process. The actin network is shaped by a plethora of actin-binding proteins; however, there is still a large gap in connecting the molecular function of these proteins with their developmental effects. Here, we summarize their function and known effects on primary root growth with a focus on their high level of specialization. Light and gravity are key factors that help us understand root growth directionality. The response of the root to gravity relies on hormonal, particularly auxin, homeostasis, and the actin cytoskeleton. Actin is necessary for the perception of the gravity stimulus via the repositioning of sedimenting statoliths, but it is also involved in mediating the growth response via the trafficking of auxin transporters and cell elongation. Furthermore, auxin and auxin analogs can affect the composition of the actin network, indicating a potential feedback loop. Light, in its turn, affects actin organization and hence, root growth, although its precise role remains largely unknown. Recently, fundamental studies with the latest techniques have given us more in-depth knowledge of the role and organization of actin in the coordination of root growth; however, there remains a lot to discover, especially in how actin organization helps cell shaping, and therefore root growth.

Allosteric Regulation of Human Plastins

Plastins/fimbrins are conserved actin-bundling proteins contributing to motility, cytokinesis, and other cellular processes by organizing actin assemblies of strikingly different geometries as in aligned bundles and branched networks. We propose that this unique ability stems from an allosteric communication between the two actin-binding domains (ABD1/2) engaged in a tight spatial association. We found that although ABD1 binds actin first, ABD2 can bind to actin three orders of magnitude stronger if not inhibited by an equally strong allosteric engagement with ABD1. Binding of ABD1 to actin lessened the inhibition, enabling weak bundling within aligned bundles. A mutation mimicking physiologically relevant phosphorylation at the ABD1-ABD2 interface strongly reduced their association, dramatically potentiating actin cross-linking. Cryo-EM reconstruction revealed the ABD1-actin interface and enabled modeling of the plastin bridge to confirm domain separation in parallel bundles. The characteristic domain organization with a strong allosteric inhibition imposed by ABD1 on ABD2 allows plastins to tune cross-linking, contributing to the assembly and remodeling of actin assemblies with different morphological and functional properties defining the unique place of plastins in actin organization.

WTS-1/LATS regulates endocytic recycling by restraining F-actin assembly in a synergistic manner

The disruption of endosomal actin architecture negatively affects endocytic recycling. However, the underlying homeostatic mechanisms that regulate actin organization during recycling remain unclear. In this study, we identified a synergistic endosomal actin assembly restricting mechanism in C. elegans involving WTS-1/LATS kinase, which is a core component of the Hippo pathway. WTS-1 resides on the sorting endosomes and colocalizes with the actin polymerization regulator PTRN-1/CAMSAPs. We observed an increase in PTRN-1-labeled structures in WTS-1-deficient cells, indicating that WTS-1 can limit the endosomal localization of PTRN-1. Accordingly, the actin overaccumulation phenotype in WTS-1-depleted cells was mitigated by the associated PTRN-1 loss. We further demonstrated that recycling defects and actin overaccumulation in WTS-1-deficient cells were reduced by the overexpression of constitutively active UNC-60A/cofilin(S3A), which aligns with the role of LATS as a positive regulator of cofilin activity. Altogether, our data confirmed previous findings, and we proposed an additional model: WTS-1 acts alongside the UNC-60A/cofilin-mediated actin disassembly to restrict the assembly of endosomal F-actin by curbing PTRN-1 dwelling on endosomes, preserving recycling transport.

Micron-scale supramolecular myosin arrays help mediate cytoskeletal assembly at mature adherens junctions

Epithelial cells assemble specialized actomyosin structures at E-Cadherin–based cell–cell junctions, and the force exerted drives cell shape change during morphogenesis. The mechanisms that build this supramolecular actomyosin structure remain unclear. We used ZO-knockdown MDCK cells, which assemble a robust, polarized, and highly organized actomyosin cytoskeleton at the zonula adherens, combining genetic and pharmacologic approaches with superresolution microscopy to define molecular machines required. To our surprise, inhibiting individual actin assembly pathways (Arp2/3, formins, or Ena/VASP) did not prevent or delay assembly of this polarized actomyosin structure. Instead, as junctions matured, micron-scale supramolecular myosin arrays assembled, with aligned stacks of myosin filaments adjacent to the apical membrane, overlying disorganized actin filaments. This suggested that myosin arrays might bundle actin at mature junctions. Consistent with this idea, inhibiting ROCK or myosin ATPase disrupted myosin localization/organization and prevented actin bundling and polarization. We obtained similar results in Caco-2 cells. These results suggest a novel role for myosin self-assembly, helping drive actin organization to facilitate cell shape change.