Actin Cytoskeleton Remodeling Accompanied by Redistribution of Adhesion Proteins Drives Migration of Cells in Different EMT States.

Epithelial-mesenchymal transition (EMT) is a process during which epithelial cells lose epithelial characteristics and gain mesenchymal features. Here, we used several cell models to study migratory activity and redistribution of cell-cell adhesion proteins in cells in different EMT states: EGF-induced EMT of epithelial IAR-20 cells; IAR-6-1 cells with a hybrid epithelial-mesenchymal phenotype; and their more mesenchymal derivatives, IAR-6-1-DNE cells lacking adherens junctions. In migrating cells, the cell-cell adhesion protein α-catenin accumulated at the leading edges along with ArpC2/p34 and α-actinin. Suppression of α-catenin shifted cell morphology from fibroblast-like to discoid and attenuated cell migration. Expression of exogenous α-catenin in MDA-MB-468 cells devoid of α-catenin drastically increased their migratory capabilities. The Y654 phosphorylated form of ß-catenin was detected at integrin adhesion complexes (IACs). Co-immunoprecipitation studies indicated that α-catenin and pY654-ß-catenin were associated with IAC proteins: vinculin, zyxin, and α-actinin. Taken together, these data suggest that in cells undergoing EMT, catenins not participating in assembly of adherens junctions may affect cell migration.

The concerted action of SEPT9 and EPLIN modulates the adhesion and migration of human fibroblasts.

Septins are cytoskeletal proteins that participate in cell adhesion, migration, and polarity establishment. The septin subunit SEPT9 directly interacts with the single LIM domain of epithelial protein lost in neoplasm (EPLIN), an actin-bundling protein. Using a human SEPT9 KO fibroblast cell line, we show that cell adhesion and migration are regulated by the interplay between both proteins. The low motility of SEPT9-depleted cells could be partly rescued by increased levels of EPLIN. The normal organization of actin-related filopodia and stress fibers was directly dependent on the expression level of SEPT9 and EPLIN. Increased levels of SEPT9 and EPLIN enhanced the size of focal adhesions in cell protrusions, correlating with stabilization of actin bundles. Conversely, decreased levels had the opposite effect. Our work thus establishes the interaction between SEPT9 and EPLIN as an important link between the septin and the actin cytoskeleton, influencing cell adhesion, motility, and migration.

PCDH17 restricts dendritic spine morphogenesis by regulating ROCK2-dependent control of the actin cytoskeleton, modulating emotional behavior.

Proper regulation of synapse formation and elimination is critical for establishing mature neuronal circuits and maintaining brain function. Synaptic abnormalities, such as defects in the density and morphology of postsynaptic dendritic spines, underlie the pathology of various neuropsychiatric disorders. Protocadherin 17 (PCDH17) is associated with major mood disorders, including bipolar disorder and depression. However, the molecular mechanisms by which PCDH17 regulates spine number, morphology, and behavior remain elusive. In this study, we found that PCDH17 functions at postsynaptic sites, restricting the number and size of dendritic spines in excitatory neurons. Selective overexpression of PCDH17 in the ventral hippocampal CA1 results in spine loss and anxiety- and depression-like behaviors in mice. Mechanistically, PCDH17 interacts with actin-relevant proteins and regulates actin filament (F-actin) organization. Specifically, PCDH17 binds to ROCK2, increasing its expression and subsequently enhancing the activity of downstream targets such as LIMK1 and the phosphorylation of cofilin serine-3 (Ser3). Inhibition of ROCK2 activity with belumosudil (KD025) ameliorates the defective F-actin organization and spine structure induced by PCDH17 overexpression, suggesting that ROCK2 mediates the effects of PCDH17 on F-actin content and spine development. Hence, these findings reveal a novel mechanism by which PCDH17 regulates synapse development and behavior, providing pathological insights into the neurobiological basis of mood disorders.

Presynaptic structural and functional plasticity are coupled by convergent Rap1 signaling.

Dynamic presynaptic actin remodeling drives structural and functional plasticity at synapses, but the underlying mechanisms remain largely unknown. Previous work has shown that actin regulation via Rac1 guanine exchange factor (GEF) Vav signaling restrains synaptic growth via bone morphogenetic protein (BMP)-induced receptor macropinocytosis and mediates synaptic potentiation via mobilization of reserve pool vesicles in presynaptic boutons. Here, we find that Gef26/PDZ-GEF and small GTPase Rap1 signaling couples the BMP-induced activation of Abelson kinase to this Vav-mediated macropinocytosis. Moreover, we find that adenylate cyclase Rutabaga (Rut) signaling via exchange protein activated by cAMP (Epac) drives the mobilization of reserve pool vesicles during post-tetanic potentiation (PTP). We discover that Rap1 couples activation of Rut-cAMP-Epac signaling to Vav-mediated synaptic potentiation. These findings indicate that Rap1 acts as an essential, convergent node for Abelson kinase and cAMP signaling to mediate BMP-induced structural plasticity and activity-induced functional plasticity via Vav-dependent regulation of the presynaptic actin cytoskeleton.

A step-by-step guide to fragmenting bundled actin filaments.

There has long been conflicting evidence as to how bundled actin filaments, found in cellular structures such as filopodia, are disassembled. In this issue, Chikireddy et al. (https://doi.org/10.1083/jcb.202312106) provide a detailed in vitro analysis of the steps involved in fragmentation of fascin-bundled actin filaments and propose a novel mechanism for severing two-filament bundles.

The amoeboid migration of monocytes in confining channels requires the local remodeling of the cortical actin cytoskeleton by cofilin-1.

Within the bloodstream, monocytes must traverse the microvasculature to prevent leukostasis, which is the entrapment of monocytes within the confines of the microvasculature. Using the model cell line, THP-1, and VCAM-1 coated channels to simulate the microvasculature surface, we demonstrate that monocytes predominantly adopt an amoeboid phenotype, which is characterized by the formation of blebs. As opposed to cortical actin flow in leader blebs, cell movement is correlated with myosin contraction at the cell rear. It was previously documented that cofilin-1 promotes cortical actin turnover at leader bleb necks in melanoma cells. In monocytes, our data suggest that cofilin-1 promotes the local upregulation of myosin contractility through actin cytoskeleton remodeling. In support of this concept, cofilin-1 is found to localize to a single cell edge. Moreover, the widespread upregulation of myosin contractility was found to inhibit migration. Thus, monocytes within the microvasculature may avoid entrapment by adopting an amoeboid mode of migration.

Prolonged depletion of profilin 1 or F-actin causes an adaptive response in microtubules.

In addition to its well-established role in actin assembly, profilin 1 (PFN1) has been shown to bind to tubulin and alter microtubule growth. However, whether PFN1's predominant control over microtubules in cells occurs through direct regulation of tubulin or indirectly through the polymerization of actin has yet to be determined. Here, we manipulated PFN1 expression, actin filament assembly, and actomyosin contractility and showed that reducing any of these parameters for extended periods of time caused an adaptive response in the microtubule cytoskeleton, with the effect being significantly more pronounced in neuronal processes. All the observed changes to microtubules were reversible if actomyosin was restored, arguing that PFN1's regulation of microtubules occurs principally through actin. Moreover, the cytoskeletal modifications resulting from PFN1 depletion in neuronal processes affected microtubule-based transport and mimicked phenotypes that are linked to neurodegenerative disease. This demonstrates how defects in actin can cause compensatory responses in other cytoskeleton components, which in turn significantly alter cellular function.

A Magnetic Pincher for the Dynamic Measurement of the Actin Cortex Thickness in Live Cells.

The actin cortex is an essential element of the cytoskeleton allowing cells to control and modify their shape. It is involved in cell division and migration. However, probing precisely the physical properties of the actin cortex has proved to be challenging: it is a thin and dynamic material, and its location in the cell-directly under the plasma membrane-makes it difficult to study with standard light microscopy and cell mechanics techniques. In this chapter, we present a novel protocol to probe dynamically the thickness of the cortex and its fluctuations using superparamagnetic microbeads in a uniform magnetic field. A bead ingested by the cell and another outside the cell attract each other due to dipolar forces. By tracking their position with nanometer precision, one can measure the thickness of the cortex pinched between two beads and monitor its evolution in time. We first present the set of elements necessary to realize this protocol: a magnetic field generator adapted to a specific imaging setup and the aforementioned superparamagnetic microbeads. Then we detail the different steps of a protocol that can be used on diverse cell types, adherent or not.

Proteomic Analysis of Prehypertensive and Hypertensive Patients: Exploring the Role of the Actin Cytoskeleton.

Hypertension is a pervasive and widespread health condition that poses a significant risk factor for cardiovascular disease, which includes conditions such as heart attack, stroke, and heart failure. Despite its widespread occurrence, the exact cause of hypertension remains unknown, and the mechanisms underlying the progression from prehypertension to hypertension require further investigation. Recent proteomic studies have shown promising results in uncovering potential biomarkers related to disease development. In this study, serum proteomic data collected from Qatar Biobank were analyzed to identify altered protein expression between individuals with normal blood pressure, prehypertension, and hypertension and to elucidate the biological pathways contributing to this disease. The results revealed a cluster of proteins, including the SRC family, CAMK2B, CAMK2D, TEC, GSK3, VAV, and RAC, which were markedly upregulated in patients with hypertension compared to those with prehypertension (fold change ≥ 1.6 or ≤-1.6, area under the curve ≥ 0.8, and q-value < 0.05). Pathway analysis showed that the majority of these proteins play a role in actin cytoskeleton remodeling. Actin cytoskeleton reorganization affects various biological processes that contribute to the maintenance of blood pressure, including vascular tone, endothelial function, cellular signaling, inflammation, fibrosis, and mechanosensing. Therefore, the findings of this study suggest a potential novel role of actin cytoskeleton-related proteins in the progression from prehypertension to hypertension. The present study sheds light on the underlying pathological mechanisms involved in hypertension and could pave the way for new diagnostic and therapeutic approaches for the treatment of this disease.

Nanoscale detection of carbon dots-induced changes in actin skeleton of neural cells.

Understanding the cytotoxicity of fluorescent carbon dots (CDs) is crucial for their applications, and various biochemical assays have been used to study the effects of CDs on cells. Knowledge on the effects of CDs from a biophysical perspective is integral to the recognition of their cytotoxicity, however the related information is very limited. Here, we report that atomic force microscopy (AFM) can be used as an effective tool for studying the effects of CDs on cells from the biophysical perspective. We achieve this by integrating AFM-based nanomechanics with AFM-based imaging. We demonstrate the performance of this method by measuring the influence of CDs on living human neuroblastoma (SH-SY5Y) cells at the single-cell level. We find that high-dose CDs can mechanically induce elevated normalized hysteresis (energy dissipation during the cell deformation) and structurally impair actin skeleton. The nanomechanical change highly correlates with the alteration of actin filaments, indicating that CDs-induced changes in SH-SY5Y cells are revealed in-depth from the AFM-based biophysical aspect. We validate the reliability of the biophysical observations using conventional biological methods including cell viability test, fluorescent microscopy, and western blot assay. Our work contributes new and significant information on the cytotoxicity of CDs from the biophysical perspective.

Myosin XI, a model of its conserved role in plant cell tip growth.

In eukaryotic cells, organelle and vesicle transport, positioning, and interactions play crucial roles in cytoplasmic organization and function. These processes are governed by intracellular trafficking mechanisms. At the core of that trafficking, the cytoskeleton and directional transport by motor proteins stand out as its key regulators. Plant cell tip growth is a well-studied example of cytoplasm organization by polarization. This polarization, essential for the cell's function, is driven by the cytoskeleton and its associated motors. This review will focus on myosin XI, a molecular motor critical for vesicle trafficking and polarized plant cell growth. We will center our discussion on recent data from the moss Physcomitrium patens and the liverwort Marchantia polymorpha. The biochemical properties and structure of myosin XI in various plant species are discussed, highlighting functional conservation across species. We further explore this conservation of myosin XI function in the process of vesicle transport in tip-growing cells. Existing evidence indicates that myosin XI actively organizes actin filaments in tip-growing cells by a mechanism based on vesicle clustering at their tips. A hypothetical model is presented to explain the essential function of myosin XI in polarized plant cell growth based on vesicle clustering at the tip. The review also provides insight into the in vivo localization and dynamics of myosin XI, emphasizing its role in cytosolic calcium regulation, which influences the polymerization of F-actin. Lastly, we touch upon the need for additional research to elucidate the regulation of myosin function.

Zyxin is important for the stability and function of podocytes, especially during mechanical stretch.

Podocyte detachment due to mechanical stress is a common issue in hypertension-induced kidney disease. This study highlights the role of zyxin for podocyte stability and function. We have found that zyxin is significantly up-regulated in podocytes after mechanical stretch and relocalizes from focal adhesions to actin filaments. In zyxin knockout podocytes, we found that the loss of zyxin reduced the expression of vinculin and VASP as well as the expression of matrix proteins, such as fibronectin. This suggests that zyxin is a central player in the translation of mechanical forces in podocytes. In vivo, zyxin is highly up-regulated in patients suffering from diabetic nephropathy and in hypertensive DOCA-salt treated mice. Furthermore, zyxin loss in mice resulted in proteinuria and effacement of podocyte foot processes that was measured by super resolution microscopy. This highlights the essential role of zyxin for podocyte maintenance in vitro and in vivo, especially under mechanical stretch.

Myosin's powerstroke transitions define atomic scale movement of cardiac thin filament tropomyosin.

Dynamic interactions between the myosin motor head on thick filaments and the actin molecular track on thin filaments drive the myosin-crossbridge cycle that powers muscle contraction. The process is initiated by Ca2+ and the opening of troponin-tropomyosin-blocked myosin-binding sites on actin. The ensuing recruitment of myosin heads and their transformation from pre-powerstroke to post-powerstroke conformation on actin produce the force required for contraction. Cryo-EM-based atomic models confirm that during this process, tropomyosin occupies three different average positions on actin. Tropomyosin pivoting on actin away from a TnI-imposed myosin-blocking position accounts for part of the Ca2+ activation observed. However, the structure of tropomyosin on thin filaments that follows pre-powerstroke myosin binding and its translocation during myosin's pre-powerstroke to post-powerstroke transition remains unresolved. Here, we approach this transition computationally in silico. We used the myosin helix-loop-helix motif as an anchor to dock models of pre-powerstroke cardiac myosin to the cleft between neighboring actin subunits along cardiac thin filaments. We then performed targeted molecular dynamics simulations of the transition between pre- and post-powerstroke conformations on actin in the presence of cardiac troponin-tropomyosin. These simulations show Arg 369 and Glu 370 on the tip of myosin Loop-4 encountering identically charged residues on tropomyosin. The charge repulsion between residues causes tropomyosin translocation across actin, thus accounting for the final regulatory step in the activation of the thin filament, and, in turn, facilitating myosin movement along the filament. We suggest that during muscle activity, myosin-induced tropomyosin movement is likely to result in unencumbered myosin head interactions on actin at low-energy cost.

Dynamic interplay of microtubule and actomyosin forces drive tissue extension.

In order to shape a tissue, individual cell-based mechanical forces have to be integrated into a global force pattern. Over the last decades, the importance of actomyosin contractile arrays, which are the key constituents of various morphogenetic processes, has been established for many tissues. Recent studies have demonstrated that the microtubule cytoskeleton mediates folding and elongation of the epithelial sheet during Drosophila morphogenesis, placing microtubule mechanics on par with actin-based processes. While these studies establish the importance of both cytoskeletal systems during cell and tissue rearrangements, a mechanistic understanding of their functional hierarchy is currently missing. Here, we dissect the individual roles of these two key generators of mechanical forces during epithelium elongation in the developing Drosophila wing. We show that wing extension, which entails columnar-to-cuboidal cell shape remodeling in a cell-autonomous manner, is driven by anisotropic cell expansion caused by the remodeling of the microtubule cytoskeleton from apico-basal to planarly polarized. Importantly, cell and tissue elongation is not associated with Myosin activity. Instead, Myosin II exhibits a homeostatic role, as actomyosin contraction balances polarized microtubule-based forces to determine the final cell shape. Using a reductionist model, we confirm that pairing microtubule and actomyosin-based forces is sufficient to recapitulate cell elongation and the final cell shape. These results support a hierarchical mechanism whereby microtubule-based forces in some epithelial systems prime actomyosin-generated forces.

Insights into the Mechanism of Action of the Degraded Limonoid Prieurianin.

Limonoids are extremely diversified in plants, with many categories of products bearing an intact, rearranged or fragmented oxygenated scaffold. A specific subgroup of fragmented or degraded limonoids derives from the tetranortriterpenoid prieurianin, initially isolated from the tree Trichilia prieuriana but also found in other plants of the Meliaceae family, including the more abundant species Aphanamixis polystachya. Prieurianin-type limonoids include about seventy compounds, among which are dregeanin and rohitukin. Prieurianin and analogs exhibit insecticidal, antimicrobial, antiadipogenic and/or antiparasitic properties but their mechanism of action remains ill-defined at present. Previous studies have shown that prieurianin, initially known as endosidin 1, stabilizes the actin cytoskeleton in plant and mammalian cells via the modulation of the architecture and dynamic of the actin network, most likely via interference with actin-binding proteins. A new mechanistic hypothesis is advanced here based on the recent discovery of the targeting of the chaperone protein Hsp47 by the fragmented limonoid fraxinellone. Molecular modeling suggested that prieurianin and, to a lesser extent dregeanin, can form very stable complexes with Hsp47 at the protein-collagen interface. Hsp-binding may account for the insecticidal action of the product. The present review draws up a new mechanistic portrait of prieurianin and provides an overview of the pharmacological properties of this atypical limonoid and its chemical family.

Mechanical interaction of myosin and native thin filament in the disused rat soleus muscle.

The disuse of skeletal limb muscles occurs in a variety of conditions, yet our comprehension of the molecular mechanisms involved in adaptation to disuse remains incomplete. We studied the mechanical characteristics of actin-myosin interaction using an in vitro motility assay and isoform composition of myosin heavy and light chains by dint of SDS-PAGE in soleus muscle of both control and hindlimb-unloaded rats. 14 days of hindlimb unloading led to the increased maximum sliding velocity of actin, reconstituted, and native thin filaments over rat soleus muscle myosin by 24 %, 19 %, and 20 %, respectively. The calcium sensitivity of the "pCa-velocity" relationship decreased. There was a 26 % increase in fast myosin heavy chain IIa (MHC IIa), a 22 % increase in fast myosin light chain 2 (MLC 2f), and a 13 % increase in fast MLC 1f content. The content of MLC 1s/v, typical for slow skeletal muscles and cardiac ventricles did not change. At the same time, MLC 1s, typical only for slow skeletal muscles, disappeared. The maximum velocity of soleus muscle native thin filaments was 24 % higher compared to control ones sliding over the same rabbit myosin. Therefore, both myosin and native thin filament kinetics could influence the mechanical characteristics of the soleus muscle. Additionally, the MLC 1s and MLC 1s/v ratio may contribute to the mechanical characteristics of slow skeletal muscle, along with MHC, MLC 2, and MLC 1 slow/fast isoforms ratio.

[Advances in the study of the actin nucleation factor Spire].

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.

Kinetic trapping organizes actin filaments within liquid-like protein droplets.

Several actin-binding proteins (ABPs) phase separate to form condensates capable of curating the actin network shapes. Here, we use computational modeling to understand the principles of actin network organization within VASP condensate droplets. Our simulations reveal that the different actin shapes, namely shells, rings, and mixture states are highly dependent on the kinetics of VASP-actin interactions, suggesting that they arise from kinetic trapping. Specifically, we show that reducing the residence time of VASP on actin filaments reduces degree of bundling, thereby promoting assembly of shells rather than rings. We validate the model predictions experimentally using a VASP-mutant with decreased bundling capability. Finally, we investigate the ring opening within deformed droplets and found that the sphere-to-ellipsoid transition is favored under a wide range of filament lengths while the ellipsoid-to-rod transition is only permitted when filaments have a specific range of lengths. Our findings highlight key mechanisms of actin organization within phase-separated ABPs.

BioID Analysis of Actin-Binding Proteins.

Proteins often exist and function as part of higher-order complexes or networks. A challenge is to identify the universe of proximal and interacting partners for a given protein. We describe how the high-activity promiscuous biotin ligase called TurboID is fused to the actin-binding peptide LifeAct to label by biotinylation proteins that bind, or are in close proximity, to actin. The rapid enzyme kinetics of TurboID allows the profiles of actin-binding proteins to be compared under different conditions, such as acute disruption of filamentous actin structures with cytochalasin D.

Molecular mechanism of actin filament elongation by formins.

Formins control the assembly of actin filaments (F-actin) that drive cell morphogenesis and motility in eukaryotes. However, their molecular interaction with F-actin and their mechanism of action remain unclear. In this work, we present high-resolution cryo-electron microscopy structures of F-actin barbed ends bound by three distinct formins, revealing a common asymmetric formin conformation imposed by the filament. Formation of new intersubunit contacts during actin polymerization sterically displaces formin and triggers its translocation. This "undock-and-lock" mechanism explains how actin-filament growth is coordinated with formin movement. Filament elongation speeds are controlled by the positioning and stability of actin-formin interfaces, which distinguish fast and slow formins. Furthermore, we provide a structure of the actin-formin-profilin ring complex, which resolves how profilin is rapidly released from the barbed end during filament elongation.