Actin filament dynamics in the actomyosin VI complex is regulated allosterically by calcium-calmodulin light chain

The contractile and enzymatic activities of myosin VI are regulated by calcium binding to associated calmodulin (CaM) light chains. We have used transient phosphorescence anisotropy to monitor the microsecond rotational dynamics of erythrosin-iodoacetamide-labeled actin with strongly bound myosin VI (MVI) and to evaluate the effect of MVI-bound CaM light chain on actin filament dynamics. MVI binding lowers the amplitude but accelerates actin filament microsecond dynamics in a Ca²⁺- and CaM-dependent manner, as indicated from an increase in the final anisotropy and a decrease in the correlation time of transient phosphorescence anisotropy decays. MVI with bound apo-CaM or Ca²⁺-CaM weakly affects actin filament microsecond dynamics, relative to other myosins (e.g., muscle myosin II and myosin Va). CaM dissociation from bound MVI damps filament rotational dynamics (i.e., increases the torsional rigidity), such that the perturbation is comparable to that induced by other characterized myosins. Analysis of individual actin filament shape fluctuations imaged by fluorescence microscopy reveals a correlated effect on filament bending mechanics. These data support a model in which Ca²⁺-dependent CaM binding to the IQ domain of MVI is linked to an allosteric reorganization of the actin binding site(s), which alters the structural dynamics and the mechanical rigidity of actin filaments. Such modulation of filament dynamics may contribute to the Ca²⁺- and CaM-dependent regulation of myosin VI motility and ATP utilization.     

Modulation of actomyosin contractility by myosin light chain phosphorylation/dephosphorylation through Rho GTPases signaling specifies axon formation in neurons

Actin depolymerization through Rho GTPases or exogenous mechanical tension has been suggested as a key determinant for the first step of neuronal polarization, the axonogenesis, in which one of the neurites starts to grow becoming the axon. The underlying mechanism and the relationship between two forces in the cells, however, are mostly unknown. Here, we report that the myosin-dependent contractility is a common effector between two forces and a critical determinant in axonogenesis and neuronal polarization. We have found that inhibition of myosin ATPase activity and modulation of myosin light chain phosphorylation/dephosphorylation through Rho GTPases signaling induced multiple axons. Moreover, overexpression of wild-type myosin light chain kinase dramatically increased filopodial structures and produced multi-axonal structures. Our results suggest that MLC phosphorylation/dephosphorylation through Rho GTPases signaling modulates the actomyosin contractility, and then in turn provides a physiological tension in neurons to induce axon.     

Multiscale morphogenesis of the mouse blastocyst by actomyosin contractility

During preimplantation development, the mouse embryo forms the blastocyst, which consists of a squamous epithelium enveloping a fluid-filled lumen and a cluster of pluripotent cells. The shaping of the blastocyst into its specific architecture is a prerequisite to implantation and further development of the embryo. Recent studies identified the central role of the actomyosin cortex in generating the forces driving the successive steps of blastocyst morphogenesis. As seen in other developing animals, actomyosin functions across spatial scales from the subcellular to the tissue levels. In addition, the slow development of the mouse embryo reveals that actomyosin contractility operates at multiple timescales with periodic cortical waves of contraction every ∼80 s and tissue remodeling over hours.     

Presenilin-dependent receptor processing is required for axon guidance

The Alzheimer's disease-linked gene presenilin is required for intramembrane proteolysis of amyloid-β precursor protein, contributing to the pathogenesis of neurodegeneration that is characterized by loss of neuronal connections, but the role of Presenilin in establishing neuronal connections is less clear. Through a forward genetic screen in mice for recessive genes affecting motor neurons, we identified the Columbus allele, which disrupts motor axon projections from the spinal cord. We mapped this mutation to the Presenilin-1 gene. Motor neurons and commissural interneurons in Columbus mutants lacking Presenilin-1 acquire an inappropriate attraction to Netrin produced by the floor plate because of an accumulation of DCC receptor fragments within the membrane that are insensitive to Slit/Robo silencing. Our findings reveal that Presenilin-dependent DCC receptor processing coordinates the interplay between Netrin/DCC and Slit/Robo signaling. Thus, Presenilin is a key neural circuit builder that gates the spatiotemporal pattern of guidance signaling, thereby ensuring neural projections occur with high fidelity.     

Actin stress fiber retraction and aggresome formation is a common cellular response to actin toxins

F-actin-stabilizing drugs induce actin aggresome formation. In this study, we found that an actin-depolymerizing drug, latrunculin A (LatA), induced actin aggresomes. Actin stress fibers were retracted and disappeared in minutes, but a large aggresome formed in consequence of LatA treatment. Because cytochalasin D and mycalolide also induced aggresome formation, these results suggest that actin aggresome formation is a common cellular response to actin toxins.     

MAP Kinase driven actomyosin rearrangement is a crucial regulator of monocyte to macrophage differentiation

Rearrangement of actin cytoskeleton correlates significantly with the immune responses as the perturbation of cytoskeletal dynamics leads to many immune deficiencies. Mechanistic insights into this correlation remain unknown. Cellular spreading, the most characteristic phenotype associated with monocyte to macrophage differentiation, led us to investigate the contribution of actomyosin dynamics in monocyte differentiation. Our observation revealed that actomyosin reorganization intrinsically governs the process of monocyte to macrophage differentiation. Further, we established that the MAPK-driven signaling pathways regulate the cellular actomyosin dynamics that direct monocyte to macrophage differentiation. We also identified P42/44 Mitogen-Activated Protein Kinase (P42/44 MAPK), P38 Mitogen-Activated Protein Kinase (P38 MAPK), MAP Kinase Activated Protein Kinase 2 (MK-2), Heat Shock Protein 27 (Hsp-27), Lim Kinase (Lim K), non-muscle cofilin (n-cofilin), Myosin Light Chain Kinase (MLCK) and Myosin Light Chain (MLC) as critical components of the signaling network. Moreover, we have shown the involvement of the same signaling cascade in 3D gel-like microenvironment induced spontaneous monocyte to macrophage differentiation and in human blood-derived PBMC differentiation. Our study reveals new mechanistic insights into the process of monocyte to macrophage differentiation.     

Differential neurite outgrowth is required for axon specification by cultured hippocampal neurons

Formation of an axon is the first morphological evidence of neuronal polarization, visible as a profound outgrowth of the axon compared with sibling neurites. One unsolved question on the mechanism of axon formation is the role of axon outgrowth in axon specification. This question was difficult to assess, because neurons freely extend their neurites in a conventional culture. Here, we leveraged surface nano/micro‐modification techniques to fabricate a template substrate for constraining neurite lengths of cultured neurons. Using the template, we asked (i) Do neurons polarize even if all neurites cannot grow sufficiently long? (ii) Would the neurite be fated to become an axon if only one was allowed to grow long? A pattern with symmetrical short paths (20 μm) was used to address the former question, and an asymmetrical pattern with one path extended to 100 μm for the latter. Axon formation was evaluated by tau‐1/MAP2 immunostaining and live‐cell imaging of constitutively‐active kinesin‐1. We found that (1) neurons cannot polarize when extension of all neurites is restricted and that (2) when only a single neurite is permitted to grow long, neurons polarize and the longest neurite becomes the axon. These results provide clear evidence that axon outgrowth is required for its specification.     

Zebrafish topped is required for ventral motor axon guidance

Zebrafish primary motor axons extend along stereotyped pathways innervating distinct regions of the developing myotome. During development, these axons make stereotyped projections to ventral and dorsal myotome regions. Caudal primary motoneurons, CaPs, pioneer axon outgrowth along ventral myotomes; whereas, middle primary motoneurons, MiPs, extend axons along dorsal myotomes. Although the development and axon outgrowth of these motoneurons has been characterized, cues that determine whether axons will grow dorsally or ventrally have not been identified. The topped mutant was previously isolated in a genetic screen designed to uncover mutations that disrupt primary motor axon guidance. CaP axons in topped mutants fail to enter the ventral myotome at the proper time, stalling at the nascent horizontal myoseptum, which demarcates dorsal from ventral axial muscle. Later developing secondary motor nerves are also delayed in entering the ventral myotome whereas all other axons examined, including dorsally projecting MiP motor axons, are unaffected in topped mutants. Genetic mosaic analysis indicates that Topped function is non-cell autonomous for motoneurons, and when wild-type cells are transplanted into topped mutant embryos, ventromedial fast muscle are the only cell type able to rescue the CaP axon defect. These data suggest that Topped functions in the ventromedial fast muscle and is essential for motor axon outgrowth into the ventral myotome.     

Robo2 is required for Slit-mediated intraretinal axon guidance

The developing optic pathway has proven one of the most informative model systems for studying mechanisms of axon guidance. The first step in this process is the directed extension of retinal ganglion cell (RGC) axons within the optic fibre layer (OFL) of the retina towards their exit point from the eye, the optic disc. Previously, we have shown that the inhibitory guidance molecules, Slit1 and Slit2, regulate two distinct aspects of intraretinal axon guidance in a region-specific manner. Using knockout mice, we have found that both of these guidance activities are mediated via Robo2. Of the four vertebrate Robos, only Robo1 and Robo2 are expressed by RGCs. In mice lacking robo1 intraretinal axon guidance occurs normally. However, in mice lacking robo2 RGC axons make qualitatively and quantitatively identical intraretinal pathfinding errors to those reported previously in Slit mutants. This demonstrates clearly that, as in other regions of the optic pathway, Robo2 is the major receptor required for intraretinal axon guidance. Furthermore, the results suggest strongly that redundancy with other guidance signals rather than different receptor utilisation is the most likely explanation for the regional specificity of Slit function during intraretinal axon pathfinding.     

Canonical wnt signaling is required for commissural axon guidance

Morphogens have been identified as guidance cues for postcrossing commissural axons in the spinal cord. Shh has a dual effect on postcrossing commissural axons: a direct repellent effect mediated by Hhip as a receptor, and an indirect effect by shaping a Wnt activity gradient. Wnts were shown to be attractants for postcrossing commissural axons in both chicken and mouse embryos. In mouse, the effects of Wnts on axon guidance were concluded to depend on the planar cell polarity (PCP) pathway. Canonical Wnt signaling was excluded based on the absence of axon guidance defects in mice lacking Lrp6 which is an obligatory coreceptor for Fzd in canonical Wnt signaling. In the loss‐of‐function studies reported here, we confirmed a role for the PCP pathway in postcrossing commissural axon guidance also in the chicken embryo. However, taking advantage of the precise temporal control of gene silencing provided by in ovo RNAi, we demonstrate that canonical Wnt signaling is also required for proper guidance of postcrossing commissural axons in the developing spinal cord. Thus, axon guidance does not seem to depend on any one of the classical Wnt signaling pathways but rather involve a network of Wnt receptors and downstream components. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 190–208, 2016     

Regulation of actomyosin contractility by PI3K in sensory axons

Phosphatidylinositol 3-kinase (PI3K) activity is known to be required for the extension of embryonic sensory axons. Inhibition of PI3K has also been shown to mediate axon retraction and growth cone collapse in response to semaphorin 3A. However, the effects of inhibiting PI3K on the neuronal cytoskeleton are not well characterized. We have previously reported that semaphorin 3A-induced axon retraction involves activation of myosin II, the formation of an intra-axonal F-actin bundle cytoskeleton, and blocks the formation of F-actin patches that serve as precursors to filopodial formation in axons. We now report that inhibition of PI3K results in activation of myosin II in axons. Inhibition of myosin II activity, or its upstream regulatory kinase RhoA-kinase, blocked axon retraction induced by inhibition of PI3K. In addition, inhibition of PI3K also induced intra-axonal F-actin bundles, which likely serve as a substratum for myosin II-based force generation during axon retraction. In axons, filopodia are formed from axonal F-actin patch precursors. Analysis of axonal F-actin patch formation in eYFP-actin expressing neurons revealed that inhibition of PI3K blocked formation of axonal F-actin patches, and thus filopodial formation. These data provide insights into the regulation of the neuronal cytoskeleton by PI3K and are consistent with the notion that decreased levels of PI3K activity mediate axon retraction and growth cone collapse in response to semaphorin 3A.     

Prior increase in metallothionein levels is required to prevent doxorubicin cardiotoxicity

Many studies have shown that metallothionein (MT) can be increased significantly by different oxidative insults in multiple organ systems. However, the increase in MT production often fails to protect against oxidative tissue injury. On the other hand, recent studies using a cardiac-specific, MT-overexpressing, transgenic mouse model have shown that MT protects against oxidative heart injury. Thus, the present study was undertaken to test the hypothesis that prior increase in MT levels is required to prevent oxidative injury. Oxidative heart injury was induced by doxorubicin (DOX), an important anticancer drug that causes severe cardiotoxicity through oxidative stress. Cardiac-specific, MT-overexpressing, transgenic mice and wild-type (WT) FVB mice were treated with DOX at 20 mg/kg. Four days after the treatment, MT concentrations were markedly elevated in the WT mouse heart. The elevated MT concentrations were comparable with those found in the transgenic mouse heart, which did not show further MT elevation in response to DOX challenge. Severe oxidative injury occurred in the heart of WT mice, including myocardial lipid peroxidation, morphological changes as examined by electron microscopy, high levels of serum creatine kinase activity, and decreased total glutathione concentrations in the heart. However, all of these pathological changes were significantly inhibited in the MT-transgenic mice. Therefore, this study demonstrates that there is a correlation between MT induction and oxidative stress in the DOX-treated mouse heart. However, MT can protect the heart from oxidative injury only if it is present prior to induction of oxidative stress.     

Dynactin is required for transport initiation from the distal axon

Dynactin is a required cofactor for the minus-end-directed microtubule motor cytoplasmic dynein. Mutations within the highly conserved CAP-Gly domain of dynactin cause neurodegenerative disease. Here, we show that the CAP-Gly domain is necessary to enrich dynactin at the distal end of primary neurons. While the CAP-Gly domain is not required for sustained transport along the axon, we find that the distal accumulation facilitates the efficient initiation of retrograde vesicular transport from the neurite tip. Neurodegenerative disease mutations in the CAP-Gly domain prevent the distal enrichment of dynactin thereby inhibiting the initiation of retrograde transport. Thus, we propose a model in which distal dynactin is a key mediator in promoting the interaction among the microtubule, dynein motor, and cargo for the efficient initiation of transport. Mutations in?the CAP-Gly domain disrupt the formation of the?motor-cargo complex, highlighting the specific defects in axonal transport that may lead to neurodegeneration.     

Actin filament organization is required for proper cAMP-dependent activation of CFTR

Previous studieshave indicated a role of the actin cytoskeleton in the regulation ofthe cystic fibrosis transmembrane conductance regulator (CFTR) ionchannel. However, the exact molecular nature of this regulation isstill largely unknown. In this report human epithelial CFTR wasexpressed in human melanoma cells genetically devoid of the filaminhomologue actin-cross-linking protein ABP-280 [ABP()]. cAMP stimulation of ABP() cells orcells genetically rescued with ABP-280 cDNA [ABP(+)] waswithout effect on whole cell Cl currents. InABP() cells expressing CFTR, cAMP was also without effect onCl conductance. In contrast, cAMP induced a 10-foldincrease in the diphenylamine-2-carboxylate (DPC)-sensitive whole cellCl currents of ABP(+)/CFTR(+) cells. Further, incells expressing both CFTR and a truncated form of ABP-280 unable tocross-link actin filaments, cAMP was also without effect on CFTRactivation. Dialysis of ABP-280 or filamin through the patch pipette,however, resulted in a DPC-inhibitable increase in the whole cellcurrents of ABP()/CFTR(+) cells. At the single-channel level,protein kinase A plus ATP activated single Clchannels only in excised patches from ABP(+)/CFTR(+) cells.Furthermore, filamin alone also induced Cl channelactivity in excised patches of ABP()/CFTR(+) cells. The presentdata indicate that an organized actin cytoskeleton is required forcAMP-dependent activation of CFTR.

     

RhoA is required for monocyte tail retraction during transendothelial migration.

Transendothelial migration of monocytes is the process by which monocytes leave the circulatory system and extravasate through the endothelial lining of the blood vessel wall and enter the underlying tissue. Transmigration requires coordination of alterations in cell shape and adhesive properties that are mediated by cytoskeletal dynamics. We have analyzed the function of RhoA in the cytoskeletal reorganizations that occur during transmigration. By loading monocytes with C3, an inhibitor of RhoA, we found that RhoA was required for transendothelial migration. We then examined individual steps of transmigration to explore the requirement for RhoA in extravasation. Our studies showed that RhoA was not required for monocyte attachment to the endothelium nor subsequent spreading of the monocyte on the endothelial surface. Time-lapse video microscopy analysis revealed that C3-loaded monocytes also had significant forward crawling movement on the endothelial monolayer and were able to invade between neighboring endothelial cells. However, RhoA was required to retract the tail of the migrating monocyte and complete diapedesis. We also demonstrate that p160ROCK, a serine/threonine kinase effector of RhoA, is both necessary and sufficient for RhoA-mediated tail retraction. Finally, we find that p160ROCK signaling negatively regulates integrin adhesions and that inhibition of RhoA results in an accumulation of beta2 integrin in the unretracted tails.     

Neuropilin-2 is required in vivo for selective axon guidance responses to secreted semaphorins

Neuropilins are receptors for class 3 secreted semaphorins, most of which can function as potent repulsive axon guidance cues. We have generated mice with a targeted deletion in the neuropilin-2 (Npn-2) locus. Many Npn-2 mutant mice are viable into adulthood, allowing us to assess the role of Npn-2 in axon guidance events throughout neural development. Npn-2 is required for the organization and fasciculation of several cranial nerves and spinal nerves. In addition, several major fiber tracts in the brains of adult mutant mice are either severely disorganized or missing. Our results show that Npn-2 is a selective receptor for class 3 semaphorins in vivo and that Npn-1 and Npn-2 are required for development of an overlapping but distinct set of CNS and PNS projections.     

Synapse loss and axon retraction in response to local muscle degeneration

During metamorphosis in the moth, Manduca sexta, the abdominal body-wall muscle DEO1 is remodeled to form the adult muscle DE5. As the larval muscle degenerates, its motoneuron loses its end plates and retracts axon branches from the degenerating muscle. Muscle degeneration is under the control of the insect hormones, the ecdysteroids. Topical application of an ecdysteroid mimic resulted in animals that produced a localized patch of pupal cuticle. Muscle fibers underlying the patch showed a gradient of degeneration. The motoneuron showed end-plate loss and axon retraction from degenerating regions of a given fiber but maintained its fine terminal branches and end plates on intact regions. The results suggest that local steroid treatments that result in local muscle degeneration bring about a loss of synaptic contacts from regions of muscle degeneration. © 1996 John Wiley & Sons, Inc.     

CRAF autophosphorylation of serine 621 is required to prevent its proteasome-mediated degradation

The CRAF protein kinase regulates proliferative, differentiation, and survival signals from activated RAS proteins to downstream effectors, most often by inducing MEK/ERK activation. A well-established model of CRAF regulation involves RAS-mediated translocation of CRAF to the plasma membrane, where it is activated by a series of events including phosphorylation. Here we have discovered a new mode of regulation that occurs prior to this step. By creating a kinase-defective version of CRAF in mice or by use of the RAF inhibitor sorafenib, we show that CRAF must first undergo autophosphorylation of serine 621 (S621). Autophosphorylation occurs in cis, does not involve MEK/ERK activation, and is essential to ensure the correct folding and stability of the protein. In the absence of S621 phosphorylation, CRAF is degraded by the proteasome by mechanisms that do not uniquely rely on the E3 ubiquitin ligase CHIP.