Syndapin I, a Synaptic Dynamin-binding Protein that Associates with the Neural Wiskott-Aldrich Syndrome Protein

The GTPase dynamin has been clearly implicated in clathrin-mediated endocytosis of synaptic vesicle membranes at the presynaptic nerve terminal. Here we describe a novel 52-kDa protein in rat brain that binds the proline-rich C terminus of dynamin. Syndapin I (synaptic, dynamin-associated protein I) is highly enriched in brain where it exists in a high molecular weight complex. Syndapin I can be involved in multiple protein–protein interactions via a src homology 3 (SH3) domain at the C terminus and two predicted coiled-coil stretches. Coprecipitation studies and blot overlay analyses revealed that syndapin I binds the brain-specific proteins dynamin I, synaptojanin, and synapsin I via an SH3 domain-specific interaction. Coimmunoprecipitation of dynamin I with antibodies recognizing syndapin I and colocalization of syndapin I with dynamin I at vesicular structures in primary neurons indicate that syndapin I associates with dynamin I in vivo and may play a role in synaptic vesicle endocytosis. Furthermore, syndapin I associates with the neural Wiskott-Aldrich syndrome protein, an actin-depolymerizing protein that regulates cytoskeletal rearrangement. These characteristics of syndapin I suggest a molecular link between cytoskeletal dynamics and synaptic vesicle recycling in the nerve terminal.

Subcellular Localization of Myosin-V in the B16 Melanoma Cells, a Wild-type Cell Line for the dilute Gene

The discovery that the dilute gene encodes a class V myosin led to the hypothesis that this molecular motor is involved in melanosome transport and/or dendrite outgrowth in mammalian melanocytes. The present studies were undertaken to gain insight into the subcellular distribution of myosin-V in the melanoma cell line B16-F10, which is wild-type for the dilute gene. Immunofluorescence studies showed some degree of superimposed labeling of myosin-V with melanosomes that predominated at the cell periphery. A subcellular fraction highly enriched in melanosomes was also enriched in myosin-V based on Western blot analysis. Immunoelectron microscopy showed myosin-V labeling associated with melanosomes and other organelles. The stimulation of B16 cells with the α-melanocyte-stimulating hormone led to a significant increase in myosin-V expression. This is the first evidence that a cAMP signaling pathway might regulate the dilute gene expression. Immunofluorescence also showed an intense labeling of myosin-V independent of melanosomes that was observed within the dendrites and at the perinuclear region. Although the results presented herein are consistent with the hypothesis that myosin-V might act as a motor for melanosome translocation, they also suggest a broader cytoplasmic function for myosin-V, acting on other types of organelles or in cytoskeletal dynamics.

Specificity of dimer formation in tropomyosins: influence of alternatively spliced exons on homodimer and heterodimer assembly.

Tropomyosins consist of nearly 100% alpha-helix and assemble into parallel and in-register coiled-coil dimers. In vitro it has been established that nonmuscle as well as native muscle tropomyosins can form homodimers. However, a mixture of muscle alpha and beta tropomyosin subunits results in the formation of the thermodynamically more stable alpha/beta heterodimer. Although the assembly preference of the muscle tropomyosin heterodimer can be understood thermodynamically, the presence of multiple tropomyosin isoforms expressed in nonmuscle cells points toward a more complex principle for determining dimer formation. We have investigated the dimerization of rat tropomyosins in living cells by the use of epitope tagging with a 16-aa sequence of the influenza hemagglutinin. Employing transfection and immunoprecipitation techniques, we have analyzed the dimers formed by muscle and nonmuscle tropomyosins in rat fibroblasts. We demonstrate that the information for homo- versus heterodimerization is contained within the tropomyosin molecule itself and that the information for the selectivity is conferred by the alternatively spliced exons. These results have important implications for models of the regulation of cytoskeletal dynamics.

A novel requirement for C. elegans Alix/ALX-1 in RME-1-mediated membrane transport

BACKGROUND: Alix/Bro1p family proteins have recently been identified as important components of multivesicular endosomes (MVEs) and are involved in the sorting of endocytosed integral membrane proteins, interacting with components of the ESCRT complex, the unconventional phospholipid LBPA, and other known endocytosis regulators. During infection, Alix can be co-opted by enveloped retroviruses, including HIV, providing an important function during virus budding from the plasma membrane. In addition, Alix is associated with the actin cytoskeleton and might regulate cytoskeletal dynamics. RESULTS: Here we demonstrate a novel physical interaction between the only apparent Alix/Bro1p family protein in C. elegans, ALX-1, and a key regulator of receptor recycling from endosomes to the plasma membrane, called RME-1. The analysis of alx-1 mutants indicates that ALX-1 is required for the endocytic recycling of specific basolateral cargo in the C. elegans intestine, a pathway previously defined by the analysis of rme-1 mutants. The expression of truncated human Alix in HeLa cells disrupts the recycling of major histocompatibility complex class I, a known Ehd1/RME-1-dependent transport step, suggesting the phylogenetic conservation of this function. We show that the interaction of ALX-1 with RME-1 in C. elegans, mediated by RME-1/YPSL and ALX-1/NPF motifs, is required for this recycling process. In the C. elegans intestine, ALX-1 localizes to both recycling endosomes and MVEs, but the ALX-1/RME-1 interaction appears to be dispensable for ALX-1 function in MVEs and/or late endosomes. CONCLUSIONS: This work provides the first demonstration of a requirement for an Alix/Bro1p family member in the endocytic recycling pathway in association with the recycling regulator RME-1.

Dynamics of Immature Secretory Granules: Role of Cytoskeletal Elements during Transport, Cortical Restriction, and F-Actin-dependent Tethering

Secretory granules store neuropeptides and hormones and exhibit regulated exocytosis upon appropriate cellular stimulation. They are generated in the trans-Golgi network as immature secretory granules, short-lived vesicular intermediates, which undergo a complex and poorly understood maturation process. Due to their short half-life and low abundance, real-time studies of immature secretory granules have not been previously possible. We describe here a pulse/chase-like system based on the expression of a human chromogranin B-GFP fusion protein in neuroendocrine PC12 cells, which permits direct visualization of the budding of immature secretory granules and their dynamics during maturation. Live cell imaging revealed that newly formed immature secretory granules are transported in a direct and microtubule-dependent manner within a few seconds to the cell periphery. Our data suggest that the cooperative action of microtubules and actin filaments restricts immature secretory granules to the F-actin-rich cell cortex, where they move randomly and mature completely within a few hours. During this maturation period, secretory granules segregate into pools of different motility. In a late phase of maturation, 60% of secretory granules were found to be immobile and about half of these underwent F-actin-dependent tethering.

Mdx myotubes have normal excitability but show reduced contraction-relaxation dynamics.

The pathogenesis of Duchenne muscular dystrophy (DMD), characterised by lack of the cytoskeletal protein dystrophin, is not completely understood. An early event in the degenerative process of DMD muscle could be a rise in cytosolic calcium concentration. In order to investigate whether this leads to alterations of contractile behaviour, we studied the excitability and contractile properties of cultured myotubes from control (C57BL/10) and mdx mice, an animal model for DMD. The myotubes were stimulated electrically and their motion was recorded photometrically. No significant differences were found between control and mdx myotubes with respect to the following parameters: chronaxy and rheobase (0.33 +/- 0.03 ms and 23 +/- 4 V vs. 0.39 +/- 0.07 ms and 22 +/- 2 V for C57 and mdx myotubes, respectively), tetanisation frequency (a similar distribution pattern was found between 5 and 30 Hz), fatigue during tetanus (found in 35% of both types of myotubes) and post-tetanic contracture. In contrast, contraction and relaxation times were longer (P < 0.005) in mdx (36 +/- 2 and 142 +/- 13 ms, respectively) than in control myotubes (26 +/- 1 and 85 +/- 9 ms, respectively). Together with our earlier findings, these results suggest a decreased capacity for calcium removal in mdx cells leading, in particular, to alterations of muscle relaxation.

Mapping the cytoskeletal prestress

Park CY, Tambe D, Alencar AM, Trepat X, Zhou EH, Millet E, Butler JP, Fredberg JJ. Mapping the cytoskeletal prestress. Am J Physiol Cell Physiol 298: C1245-C1252, 2010. First published February 17, 2010; doi: 10.1152/ajpcell.00417.2009.-Cell mechanical properties on a whole cell basis have been widely studied, whereas local intracellular variations have been less well characterized and are poorly understood. To fill this gap, here we provide detailed intracellular maps of regional cytoskeleton (CSK) stiffness, loss tangent, and rate of structural rearrangements, as well as their relationships to the underlying regional F-actin density and the local cytoskeletal prestress. In the human airway smooth muscle cell, we used micropatterning to minimize geometric variation. We measured the local cell stiffness and loss tangent with optical magnetic twisting cytometry and the local rate of CSK remodeling with spontaneous displacements of a CSK-bound bead. We also measured traction distributions with traction microscopy and cell geometry with atomic force microscopy. On the basis of these experimental observations, we used finite element methods to map for the first time the regional distribution of intracellular prestress. Compared with the cell center or edges, cell corners were systematically stiffer and more fluidlike and supported higher traction forces, and at the same time had slower remodeling dynamics. Local remodeling dynamics had a close inverse relationship with local cell stiffness. The principal finding, however, is that systematic regional variations of CSK stiffness correlated only poorly with regional F-actin density but strongly and linearly with the regional prestress. Taken together, these findings in the intact cell comprise the most comprehensive characterization to date of regional variations of cytoskeletal mechanical properties and their determinants.

Percolation in a network with long-range connections: Implications for cytoskeletal structure and function

Cell shape, signaling, and integrity depend on cytoskeletal organization. In this study we describe the cytoskeleton as a simple network of filamentary proteins (links) anchored by complex protein structures (nodes). The structure of this network is regulated by a distance-dependent probability of link formation as P = p/d(s), where p regulates the network density and s controls how fast the probability for link formation decays with node distance (d). It was previously shown that the regulation of the link lengths is crucial for the mechanical behavior of the cells. Here we examined the ability of the two-dimensional network to percolate (i.e. to have end-to-end connectivity), and found that the percolation threshold depends strongly on s. The system undergoes a transition around s = 2. The percolation threshold of networks with s < 2 decreases with increasing system size L, while the percolation threshold for networks with s > 2 converges to a finite value. We speculate that s < 2 may represent a condition in which cells can accommodate deformation while still preserving their mechanical integrity. Additionally, we measured the length distribution of F-actin filaments from publicly available images of a variety of cell types. In agreement with model predictions, cells originating from more deformable tissues show longer F-actin cytoskeletal filaments. (C) 2008 Elsevier B.V. All rights reserved.

Dynamics of contacts between lamellae of fibroblasts: Essential role of the actin cytoskeleton

We investigated actin cytoskeletal and adhesion molecule dynamics during collisions of leading lamellae of nontransformed and oncogene-transformed fibroblasts. By using real-time video microscopy, it was found that during lamellar collision there was considerable overlapping of leading lamellae followed by subsequent retraction. Overlapping of nontransformed fibroblasts was accompanied by formation of β-catenin-positive contact structures organized into strands oriented parallel to the long axis of the cell that were associated with bundles of actin filaments. Maintenance of such cell–cell contact structures critically depended on the contractility of actin cytoskeleton, as inhibition of contractility with serum-free medium or 2,3-butanedione 2-monoxime (BDM) resulted in loss of strand formation. Strand formation was recovered when cells in serum-free medium were incubated with the microtubule inhibitor nocodazole, which is known to increase contractility. Oncogene-transformed fibroblasts reacted to collisions with responses similar to nontransformed fibroblasts but did not develop well-organized cell–cell contacts. A model is presented to describe how differences in the organization of the actin cytoskeleton could account for the structurally distinct responses to cell–cell contact by polarized fibroblastic cells versus nonpolarized epithelial cells.

Birefringence Imaging Directly Reveals Architectural Dynamics of Filamentous Actin in Living Growth ConesV⃞

We have investigated the dynamic behavior of cytoskeletal fine structure in the lamellipodium of nerve growth cones using a new type of polarized light microscope (the Pol-Scope). Pol-Scope images display with exquisite resolution and definition birefringent fine structures, such as filaments and membranes, without having to treat the cell with exogenous dyes or fluorescent labels. Furthermore, the measured birefringence of protein fibers in the thin lamellipodial region can be interpreted in terms of the number of filaments in the bundles. We confirmed that birefringent fibers are actin-based using conventional fluorescence-labeling methods. By recording movies of time-lapsed Pol-Scope images, we analyzed the creation and dynamic composition of radial fibers, filopodia, and intrapodia in advancing growth cones. The strictly quantitative information available in time-lapsed Pol-Scope images confirms previously deduced behavior and provides new insight into the architectural dynamics of filamentous actin.

Arp2/3 complex and actin dynamics are required for actin-based mitochondrial motility in yeast

The Arp2/3 complex is implicated in actin polymerization-driven movement of Listeria monocytogenes. Here, we find that Arp2p and Arc15p, two subunits of this complex, show tight, actin-independent association with isolated yeast mitochondria. Arp2p colocalizes with mitochondria. Consistent with this result, we detect Arp2p-dependent formation of actin clouds around mitochondria in intact yeast. Cells bearing mutations in ARP2 or ARC15 genes show decreased velocities of mitochondrial movement, loss of all directed movement and defects in mitochondrial morphology. Finally, we observe a decrease in the velocity and extent of mitochondrial movement in yeast in which actin dynamics are reduced but actin cytoskeletal structure is intact. These results support the idea that the movement of mitochondria in yeast is actin polymerization driven and that this movement requires Arp2/3 complex.

Cytoskeletal microdifferentiation: A mechanism for organizing morphological plasticity in dendrites

Experimental evidence suggests that microfilaments and microtubules play contrasting roles in regulating the balance between motility and stability in neuronal structures. Actin-containing microfilaments are associated with structural plasticity, both during development when their dynamic activity drives the exploratory activity of growth cones and after circuit formation when the actin-rich dendritic spines of excitatory synapses retain a capacity for rapid changes in morphology. By contrast, microtubules predominate in axonal and dendritic processes, which appear to be morphologically relatively more stable. To compare the cytoplasmic distributions and dynamics of microfilaments and microtubules we made time-lapse recordings of actin or the microtubule-associated protein 2 tagged with green fluorescent protein in neurons growing in dispersed culture or in tissue slices from transgenic mice. The results complement existing evidence indicating that the high concentrations of actin present in dendritic spines is a specialization for morphological plasticity. By contrast, microtubule-associated protein 2 is limited to the shafts of dendrites where time-lapse recordings show little evidence for dynamic activity. A parallel exists between the partitioning of microfilaments and microtubules in motile and stable domains of growing processes during development and between dendrite shafts and spines at excitatory synapses in established neuronal circuits. These data thus suggest a mechanism, conserved through development and adulthood, in which the differential dynamics of actin and microtubules determine the plasticity of neuronal structures.

Contact interactions between epitheliocytes and fibroblasts: Formation of heterotypic cadherin-containing adhesion sites is accompanied by local cytoskeletal reorganization

Contact interactions between different cell types play a number of important roles in development, for example in cell sorting, tissue organization, and ordered migration of cells. The nature of such heterocellular interactions, in contrast to interactions between cells of the same type, remains largely unknown. In this report, we present experimental data examining the dynamics of heterocellular interactions between epitheliocytes and fibroblasts, which express different cadherin cell adhesion molecules and possess different actin cytoskeletal organizations. Our analysis revealed two striking features of heterocellular contact. First, the active free edge of an epitheliocyte reorganizes its actin cytoskeleton after making contact with a fibroblast. Upon contact with the leading edge of a fibroblast, epitheliocytes disassemble their marginal bundle of actin filaments and reassemble actin filaments into a geometric organization more typical of a fibroblast lamella. Second, epitheliocytes and fibroblasts form cell–cell adhesion structures that have an irregular organization and are associated with components of cell adhesion complexes. The structural organization of these adhesions is more closely related to the type of contacts formed between fibroblasts rather than to those between epitheliocytes. Heterotypic epithelio-fibroblastic contacts, like homotypic contacts between fibroblasts, are transient and do not lead to formation of stable contact interactions. We suggest that heterocellular contact interactions in culture may be regarded as models of how tissue systems consisting of epithelia and mesenchyme interact and become organized in vivo.

Myosin dynamics in live Dictyostelium cells.

Conventional myosin plays a key role in the cytoskeletal reorganization necessary for cytokinesis, migration, and morphological changes associated with development in nonmuscle cells. We have made a fusion between the green fluorescent protein (GFP) and the Dictyostelium discoideum myosin heavy chain (GFP-myosin). The unique Dictyostelium system allows us to test the GFP-tagged myosin for activity both in vivo and in vitro. Expression of GFP-myosin rescues all myosin null cell defects. Additionally, GFP-myosin purified from these cells exhibits the same ATPase activities and in vitro motility as wild-type myosin. GFP-myosin is concentrated in the cleavage furrow during cytokinesis and in the posterior cortex of migrating cells. Surprisingly, GFP-myosin concentration increases transiently in the tips of retracting pseudopods. Contrary to previous thinking, this suggests that conventional myosin may play an important role in the dynamics of pseudopods as well as filopodia, lamellipodia, and other cellular protrusions.

Apicomplexans pulling the strings: manipulation of the host cell cytoskeleton dynamics

Invasive stages of apicomplexan parasites require a host cell to survive, proliferate and advance to the next life cycle stage. Once invasion is achieved, apicomplexans interact closely with the host cell cytoskeleton, but in many cases the different species have evolved distinct mechanisms and pathways to modulate the structural organization of cytoskeletal filaments. The host cell cytoskeleton is a complex network, largely, but not exclusively, composed of microtubules, actin microfilaments and intermediate filaments, all of which are modulated by associated proteins, and it is involved in diverse functions including maintenance of cell morphology and mechanical support, migration, signal transduction, nutrient uptake, membrane and organelle trafficking and cell division. The ability of apicomplexans to modulate the cytoskeleton to their own advantage is clearly beneficial. We here review different aspects of the interactions of apicomplexans with the three main cytoskeletal filament types, provide information on the currently known parasite effector proteins and respective host cell targets involved, and how these interactions modulate the host cell physiology. Some of these findings could provide novel targets that could be exploited for the development of preventive and/or therapeutic strategies.