Form-Finding Model Shows How Cytoskeleton Network Stiffness Is Realized

In eukaryotic cells the actin-cytoskeletal network provides stiffness and the driving force that contributes to changes in cell shape and cell motility, but the elastic behavior of this network is not well understood. In this paper a two dimensional form-finding model is proposed to investigate the elasticity of the actin filament network. Utilizing an initially random array of actin filaments and actin-cross-linking proteins the form-finding model iterates until the random array is brought into a stable equilibrium configuration. With some care given to actin filament density and length, distance between host sites for cross-linkers, and overall domain size the resulting configurations from the form-finding model are found to be topologically similar to cytoskeletal networks in real cells. The resulting network may then be mechanically exercised to explore how the actin filaments deform and align under load and the sensitivity of the network’s stiffness to actin filament density, length, etc. Results of the model are consistent with the experimental literature, e.g. actin filaments tend to re-orient in the direction of stretching; and the filament relative density, filament length, and actin-cross-linking protein’s relative density, control the actin-network stiffness. The model provides a ready means of extension to more complicated domains and a three-dimensional form-finding model is under development as well as models studying the formation of actin bundles.     

Functional cooperation between the microtubule and actin cytoskeletons

In diverse cell types, microtubule (MT) and actin filament networks cooperate functionally during a wide variety of processes, including vesicle and organelle transport, cleavage furrow placement, directed cell migration, spindle rotation, and nuclear migration. The mechanisms by which MTs and actin filaments cooperate to mediate these different processes can be grouped into two broad categories: coordinated MT- and actin-based transport to move vesicles, organelles, and cell fate determinants; and targeting and capture of MT ends at cortical actin sites. Over the past several years, a growing number of cellular factors that bridge these cytoskeletal systems have been identified. These include 'hetero-motor' complexes (physically associated myosin and kinesin), myosin-CLIP170 complexes, formin homology (FH) proteins, dynein and the dynactin complex, Kar9p, coronin, Kelch repeat-containing proteins, and ERM proteins.     

Models of motor-assisted transport of intracellular particles

One-dimensional models are presented for the macroscopic intracellular transport of vesicles and organelles by molecular motors on a network of aligned intracellular filaments. A motor-coated vesicle or organelle is described as a diffusing particle binding intermittently to filaments, when it is transported at the motor velocity. Two models are treated in detail: 1) a unidirectional model, where only one kind of motor is operative and all filaments have the same polarity; and 2) a bidirectional model, in which filaments of both polarities exist (for example, a randomly polarized actin network for myosin motors) and/or particles have plus-end and minus-end motors operating on unipolar filaments (kinesin and dynein on microtubules). The unidirectional model provides net particle transport in the absence of a concentration gradient. A symmetric bidirectional model, with equal mixtures of filament polarities or plus-end and minus-end motors of the same characteristics, provides rapid transport down a concentration gradient and enhanced dispersion of particles from a point source by motor-assisted diffusion. Both models are studied in detail as a function of the diffusion constant and motor velocity of bound particles, and their rates of binding to and detachment from filaments. These models can form the basis of more realistic models for particle transport in axons, melanophores, and the dendritic arms of melanocytes, in which networks of actin filaments and microtubules coexist and motors for both types of filament are implicated.     

Distinct cytoskeletal tracks direct individual vesicle populations to the apical membrane of epithelial cells

A key aspect in the structure of epithelial and neuronal cells is the maintenance of a polarized organization based on highly specific sorting machinery at the exit site of the trans Golgi network (TGN). Epithelial cells sort protein and lipid components into different sets of carriers for the apical or basolateral plasma membrane. The two intestinal proteins lactase-phlorizin hydrolase (LPH) and sucrase-isomaltase (SI) are delivered to the apical plasma membrane of epithelial cells with high fidelity but differ in their affinity to detergent-insoluble, glycolipid-enriched complexes (DIGs). Using a two-color labeling technique, we have recently characterized two post-Golgi vesicle populations that direct LPH and SI separately to the apical cell surface. Here, we investigated the structure and identification of protein components in these vesicle populations and assessed the role of cytoskeletal post-Golgi transport routes for apical cargo. Apart from the central role of microtubules in vesicle transport, we demonstrate that the transport of SI-carrying apical vesicles (SAVs) occurs along actin tracks in the cellular periphery, whereas LPH-carrying apical vesicles (LAVs) are transferred in an actin-independent fashion to the apical membrane. Our data further indicate that myosin 1A is the actin-associated motor protein that drives SAVs along actin filaments to the apical cell surface.     

Diffusion rate limitations in actin-based propulsion of hard and deformable particles

The mechanism by which actin polymerization propels intracellular vesicles and invasive microorganisms remains an open question. Several recent quantitative studies have examined propulsion of biomimetic particles such as polystyrene microspheres, phospholipid vesicles, and oil droplets. In addition to allowing quantitative measurement of parameters such as the dependence of particle speed on its size, these systems have also revealed characteristic behaviors such a saltatory motion of hard particles and oscillatory deformation of soft particles. Such measurements and observations provide tests for proposed mechanisms of actin-based motility. In the actoclampin filament end-tracking motor model, particle-surface-bound filament end-tracking proteins are involved in load-insensitive processive insertion of actin subunits onto elongating filament plus-ends that are persistently tethered to the surface. In contrast, the tethered-ratchet model assumes working filaments are untethered and the free-ended filaments grow as thermal ratchets in a load-sensitive manner. This article presents a model for the diffusion and consumption of actin monomers during actin-based particle propulsion to predict the monomer concentration field around motile particles. The results suggest that the various behaviors of biomimetic particles, including dynamic saltatory motion of hard particles and oscillatory vesicle deformations, can be quantitatively and self-consistently explained by load-insensitive, diffusion-limited elongation of (+)-end-tethered actin filaments, consistent with predictions of the actoclampin filament-end tracking mechanism.     

Cytoskeleton and vesicle mobility in astrocytes

Exocytotic vesicles in astrocytes are increasingly viewed as essential in astrocyte-to-neuron communication in the brain. In neurons and excitable secretory cells, delivery of vesicles to the plasma membrane for exocytosis involves an interaction with the cytoskeleton, in particular microtubules and actin filaments. Whether cytoskeletal elements affect vesicle mobility in astrocytes is unknown. We labeled single vesicles with fluorescent atrial natriuretic peptide and monitored their mobility in rat astrocytes with depolymerized microtubules, actin, and intermediate filaments and in mouse astrocytes deficient in the intermediate filament proteins glial fibrillary acidic protein and vimentin. In astrocytes, as in neurons, microtubules participated in directional vesicle mobility, and actin filaments played an important role in this process. Depolymerization of intermediate filaments strongly affected vesicle trafficking and in their absence the fraction of vesicles with directional mobility was reduced.     

Modeling myosin Va liposome transport through actin filament networks reveals a percolation threshold that modulates transport properties

Myosin Va (myoVa) motors transport membrane-bound cargo through three-dimensional, intracellular actin filament networks. We developed a coarse-grained, in silico model to predict how actin filament density (3-800 filaments) within a randomly oriented actin network affects fluid-like liposome (350 nm vs. 1750 nm) transport by myoVa motors. Five thousand simulated liposomes transported within each network adopted one of three states: transport, tug-of-war, or diffusion. Diffusion due to liposome detachment from actin rarely occurred given at least 10 motors on the liposome surface. However, with increased actin density, liposomes transitioned from primarily directed transport on single actin filaments to an apparent random walk, resulting from a mixture of transport and tug-of-wars as the probability of encountering additional actin filaments increased. This phase transition arises from a percolation phase transition at a critical number of accessible actin filaments, N_c. N_c is a geometric property of the actin network that depends only on the position and polarity of the actin filaments, transport distance, and the liposome diameter, as evidenced by a fivefold increase in liposome diameter resulting in a fivefold decrease in N_c. Thus in cells, actin network density and cargo size may be regulated to match cargo delivery to the cell’s physiological demands.     

The cytoskeletal architecture of the presynaptic terminal and molecular structure of synapsin 1

We have examined the cytoskeletal architecture and its relationship with synaptic vesicles in synapses by quick-freeze deep-etch electron microscopy (QF.DE). The main cytoskeletal elements in the presynaptic terminals (neuromuscular junction, electric organ, and cerebellar cortex) were actin filaments and microtubules. The actin filaments formed a network and frequently were associated closely with the presynaptic plasma membranes and active zones. Short, linking strands approximately 30 nm long were found between actin and synaptic vesicles, between microtubules and synaptic vesicles. Fine strands (30-60 nm) were also found between synaptic vesicles. Frequently spherical structures existed in the middle of the strands between synaptic vesicles. Another kind of strand (approximately 100 nm long, thinner than the actin filaments) between synaptic vesicles and plasma membranes was also observed. We have examined the molecular structure of synapsin 1 and its relationship with actin filaments, microtubules, and synaptic vesicles in vitro using the low angle rotary shadowing technique and QF.DE. The synapsin 1, approximately 47 nm long, was composed of a head (approximately 14 nm diam) and a tail (approximately 33 nm long), having a tadpole-like appearance. The high resolution provided by QF.DE revealed that a single synapsin 1 cross-linked actin filaments and linked actin filaments with synaptic vesicles, forming approximately 30-nm short strands. The head was on the actin and the tail was attached to the synaptic vesicle or actin filament. Microtubules were also cross-linked by a single synapsin 1, which also connected a microtubule to synaptic vesicles, forming approximately 30 nm strands. The spherical head was on the microtubules and the tail was attached to the synaptic vesicles or to microtubules. Synaptic vesicles incubated with synapsin 1 were linked with each other via fine short fibrils and frequently we identified spherical structures from which two or three fibril radiated and cross-linked synaptic vesicles. We have examined the localization of synapsin 1 using ultracryomicrotomy and colloidal gold-immunocytochemistry of anti-synapsin 1 IgG. Synapsin 1 was exclusively localized in the regions occupied by synaptic vesicles. Statistical analyses indicated that synapsin 1 is located mostly at least approximately 30 nm away from the presynaptic membrane. These data derived via three different approaches suggest that synapsin 1 could be a main element of short linkages between actin filaments and synaptic vesicles, and between microtubules and synaptic vesicles, and between synaptic vesicles in the nerve terminals.(ABSTRACT TRUNCATED AT 400 WORDS)     

An actin-dependent mechanism for long-range vesicle transport

Intracellular transport is vital for the function, survival and architecture of every eukaryotic cell. Long-range transport in animal cells is thought to depend exclusively on microtubule tracks. This study reveals an unexpected actin-dependent but microtubule-independent mechanism for long-range transport of vesicles. Vesicles organize their own actin tracks by recruiting the actin nucleation factors Spire1, Spire2 and Formin-2, which assemble an extensive actin network from the vesicles' surfaces. The network connects the vesicles with one another and with the plasma membrane. Vesicles move directionally along these connections in a myosin-Vb-dependent manner to converge and to reach the cell surface. The overall outward-directed movement of the vesicle-actin network is driven by recruitment of vesicles to the plasma membrane in the periphery of the oocyte. Being organized in a dynamic vesicle-actin network allows vesicles to move in a local random manner and a global directed manner at the same time: they can reach any position in the cytoplasm, but also move directionally to the cell surface as a collective. Thus, collective movement within a network is a powerful and flexible mode of vesicle transport.     

Membrane associated nonmuscle myosin II functions as a motor for actin-based vesicle transport in clam oocyte extracts

Nonmuscle myosin II (Myo2) has been shown to associate with membranes of the trans-Golgi network and to be involved in Golgi to ER retrograde protein transport. Here, we provide evidence that Myo2 not only associates with membranes but functions to transport vesicles on actin filaments (AFs). We used extracts from unactivated clam oocytes for these studies. AFs assembled spontaneously in these extracts and myosin-dependent vesicle transport was observed upon activation. In addition, actin bundles formed and moved relative to each other at an average speed of 0.30 microm/s. Motion analysis revealed that vesicles moved on the spontaneously assembled AFs at speeds greater than 1 microm/s. The motor on these vesicles was identified as a member of the nonmuscle Myo2 family based on sequence determination by Edman chemistry. Vesicles in these extracts were purified by sucrose gradient centrifugation and movement was reconstituted in vitro using skeletal muscle actin coated coverslips. When peripheral membrane proteins of vesicles including Myo2 were removed by salt stripping or when extracts were treated with an antibody specific to clam oocyte nonmuscle Myo2, vesicle movement was inhibited. Blebbistatin, a Myo2 specific inhibitor, also blocked vesicle movement. Myo2 light chain kinase activity was found to be essential for vesicle movement and sliding of actin bundles. Together, our data provide direct evidence that nonmuscle Myo2 is involved in actin-dependent vesicle transport in clam oocytes.     

Insulin action on GLUT4 traffic visualized in single 3T3-l1 adipocytes by using ultra-fast microscopy

A novel imaging technology, high-speed microscopy, has been used to visualize the process of GLUT4 translocation in response to insulin in single 3T3-L1 adipocytes. A key advantage of this technology is that it requires extremely low light exposure times, allowing the quasi-continuous capture of information over 20-30 min without photobleaching or photodamage. The half-time for the accumulation of GLUT4-eGFP (enhanced green fluorescent protein) at the plasma membrane in a single cell was found to be of 5-7 min at 37 degrees C. This half-time is substantially longer than that of exocytic vesicle fusion in neuroendocrine cells, suggesting that additional regulatory mechanisms are involved in the stimulation of GLUT4 translocation by insulin. Analysis of four-dimensional images (3-D over time) revealed that, in response to insulin, GLUT4-eGFP-enriched vesicles rapidly travel from the juxtanuclear region to the plasma membrane. In nontransfected adipocytes, impairment of microtubule and actin filament function inhibited insulin-stimulated glucose transport by 70 and 50%, respectively. When both filament systems were impaired insulin-stimulated glucose transport was completely inhibited. Taken together, the data suggest that the regulation of long-range motility of GLUT4-containing vesicles through the interaction with microtubule- and actin-based cytoskeletal networks plays an important role in the overall effect of insulin on GLUT4 translocation.     

Regulated Expression of Human Filaggrin in Keratinocytes Results in Cytoskeletal Disruption, Loss of Cell–Cell Adhesion, and Cell Cycle Arrest

Filaggrin is an intermediate filament (IF)-associated protein that aggregates keratin IFs in vitro and is thought to perform a similar function during the terminal differentiation of epidermal keratinocytes. To further explore the role of filaggrin in the cytoskeletal rearrangement that accompanies epidermal differentiation, we generated keratinocyte cell lines that express human filaggrin using a tetracycline-inducible promoter system. Filaggrin expression resulted in reduced keratinocyte proliferation and caused an alteration in cell cycle distribution consistent with a post-G1 phase arrest. Keratin filament distribution was disrupted in filaggrin-expressing lines, while the organization of actin microfilaments and microtubules was more mildly affected. Evidence for direct interaction of filaggrin and keratin IFs was seen by overlay assays of GFP-filaggrin with keratin proteins in vitro and by filamentous filaggrin distribution in cells with low levels of expression. Cells expressing moderate to high levels of filaggrin showed a rounded cell morphology, loss of cell-cell adhesion, and compacted cytoplasm. There was also partial or complete loss of the desmosomal proteins desmoplakin, plakoglobin, and desmogleins from cell-cell borders, while the distribution of the adherens junction protein E-cadherin was not affected. No alterations in keratin cytoskeleton, desmosomal protein distribution, or cell shape were observed in control cell lines expressing beta-galactosidase. Filaggrin altered the cell shape and disrupted the actin filament distribution in IF-deficient SW13 cells, demonstrating that filaggrin can affect cell morphology independent of the presence of a cytoplasmic IF network. These studies demonstrate that filaggrin, in addition to its known effects on IF organization, can affect the distribution of other cytoskeletal elements including actin microfilaments, which can occur in the absence of a cytoplasmic IF network. Further, filaggrin can disrupt the distribution of desmosome proteins, suggesting an additional role(s) for this protein in the cytoskeletal and desmosomal reorganization that occurs at the granular to cornified cell transition during terminal differentiation of epidermal keratinocytes.     

Myosin-V,a versatile motor for short-range vesicle transport

Myosin-V is a versatile motor involved in short-range transport of vesicles in the actin-rich cortex of the cell. It binds to several different kinds of vesicles, and the mechanism by which it interacts with the vesicle surface is being unraveled, primarily in melanocytes. Members of the Rab family of G-proteins are required for the recruitment of myosin-V to vesicles. Rab27a and its rabphilin-like effector protein, Melanophilin, recruit myosin-Va to melanosomes and appear to serve as the membrane receptor. Myosin-V is also involved in fast axonal/dendritic transport and, interestingly, it forms a complex with kinesin, a microtubule-based motor. This kinesin/myosin-V heteromotor complex allows long-range movement of vesicles within axons and dendrites on microtubules and short-range movement in the dendritic spines and axon terminals on actin filaments. The direct interaction of motors from both filament systems may represent the mechanism by which the transition of vesicles from microtubules to actin filaments is regulated .     

Myosin VI altered at threonine 406 stabilizes actin filaments in vivo

Myosin VI is a minus-end directed actin-based molecular motor implicated in uncoated endocytic vesicle transport. Recent kinetic studies have shown that myosin VI displays altered ADP release kinetics under different load conditions allowing myosin VI to serve alternately as a transporter or as an actin tether. We theorized that one potential regulatory event to modulate between these kinetic choices is phosphorylation at a conserved site, threonine 406 (T406) in the myosin VI motor domain. Alterations mimicking the phosphorylated (T406E) and dephosphorylated state (T406A) were introduced into a GFP-myosin VI fusion (GFP-M6). Live cell imaging revealed that GFP-M6(T406E) expression changed the path myosin VI took in its transport of uncoated endocytic vesicles. Rather than routing vesicles inwards as seen in GFP-M6 and GFP-M6(T406A) expressing cells, GFP-M6(T406E) moved vesicles into clusters at distinct peripheral sites. GFP-M6(T406E) expression also increased the density of the actin cytoskeleton. Filaments were enriched at the vesicle cluster sites. This was not due to a gross redistribution of the actin polymerization machinery. Instead the filament density correlated to the fixed positioning of GFP-M6(T406E)-associated vesicles on F-actin, leading to inhibition of actin depolymerization. Our study suggests that phosphorylation at T406 changes the nature of myosin VI's interaction with actin in vivo.     

Strain field in actin filament network in lamellipodia of migrating cells: Implication for network reorganization

Cell motility is spatiotemporally regulated by interactions among mechanical and biochemical factors involved in the regulation of cytoskeletal actin structure reorganization. Although the molecular mechanisms underlying cell motility have been well investigated, the contributions of mechanical factors such as strain in the network reorganization remain unclear. In this study, we have quantitatively evaluated the strain field in the actin filament network forming the lamellipodia of migrating fish keratocytes to elucidate the mechanism by which actin filament network reorganization is regulated by biomechanical factors. The results highlight the existence of a negative (compressive) strain in the lamellipodia whose direction is parallel to that of cell movement. A close correlation was found between the distributions of the strain and the actin filament density in the lamellipodia, suggesting that negative strain may be involved in filament depolymerization. Based on this result, we propose a selective depolymerization model which suggests that negative strain may couple with biomechanical factors such as ADF/cofilin to promote selective depolymerization of filaments oriented in the direction of the deformation because such filaments experience relatively higher levels of the deformation. This model, in conjunction with others, may explain the observed reduction in filament density and the reorganization of actin filament network at the back of the lamellipodia of migrating fish keratocytes. Thus, we suggest that by coupling with biochemical factors, mechanical factors are involved in the regulation of actin filament depolymerization, thereby contributing to the regulation of cell motility.     

How peptide hormone vesicles are transported to the secretion site for exocytosis

Post-Golgi transport of peptide hormone-containing vesicles from the site of genesis at the trans-Golgi network to the release site at the plasma membrane is essential for activity-dependent hormone secretion to mediate various endocrinological functions. It is known that these vesicles are transported on microtubules to the proximity of the release site, and they are then loaded onto an actin/myosin system for distal transport through the actin cortex to just below the plasma membrane. The vesicles are then tethered to the plasma membrane, and a subpopulation of them are docked and primed to become the readily releasable pool. Cytoplasmic tails of vesicular transmembrane proteins, as well as many cytosolic proteins including adaptor proteins, motor proteins, and guanosine triphosphatases, are involved in vesicle budding, the anchoring of the vesicles, and the facilitation of movement along the transport systems. In addition, a set of cytosolic proteins is also necessary for tethering/docking of the vesicles to the plasma membrane. Many of these proteins have been identified from different types of (neuro)endocrine cells. Here, we summarize the proteins known to be involved in the mechanisms of sorting various cargo proteins into regulated secretory pathway hormone-containing vesicles, movement of these vesicles along microtubules and actin filaments, and their eventual tethering/docking to the plasma membrane for hormone secretion.     

Action of cytochalasin D on cytoskeletal networks

Extraction of SC-1 cells (African green monkey kidney) with the detergent Triton X-100 in combination with stereo high-voltage electron microscopy of whole mount preparations has been used as an approach to determine the mode of action of cytochalasin D on cells. The cytoskeleton of extracted BSC-1 cells consists of substrate-associated filament bundles (stress fibers) and a highly cross-linked network of four major filament types extending throughout the cell body; 10-nm filaments, actin microfilaments, microtubules, and 2- to 3-nm filaments. Actin filaments and 2- to 3-nm filaments form numerous end- to-side contacts with other cytoskeletal filaments. Cytochalasin D treatment severely disrupts network organization, increases the number of actin filament ends, and leads to the formation of filamentous aggregates or foci composed mainly of actin filaments. Metabolic inhibitors prevent filament redistribution, foci formation, and cell arborization, but not disorganization of the three-dimensional filament network. In cells first extracted and then treated with cytochalasin D, network organization is disrupted, and the number of free filament ends is increased. Supernates of preparations treated in this way contain both short actin filaments and network fragments (i.e., actin filaments in end-to-side contact with other actin filaments). It is proposed that the dramatic effects of cytochalasin D on cells result from both a direct interaction of the drug with the actin filament component of cytoskeletal networks and a secondary cellular response. The former leads to an immediate disruption of the ordered cytoskeletal network that appears to involve breaking of actin filaments, rather than inhibition of actin filament-filament interactions (i.e., disruption of end-to-side contacts). The latter engages network fragments in an energy-dependent (contractile) event that leads to the formation of filament foci.     

Actin cytoskeletal network in aging and cancer.

The cytoskeleton is being recognized as an important modulator of metabolic functions of the cell. The actin cytoskeletal network, in particular, is involved in events regulating cell proliferation and differentiation. The state of actin in a variety of cell types is regulated by signals arising from the cell surface through a wide spectrum of interactions. In this review, we explore the role of actin cytoskeletal network in a series of events which are known to influence cell proliferation and differentiation. These include interaction of actin network with extracellular matrix proteins, cell surface membranes, second messengers, cytoplasmic enzymes and the nucleus. Because of the involvement of the actin network in such diverse interactions, we propose that alterations in the actin cytoskeletal function may be an important aspect of generalized decrease in cellular functions associated with aging. Preliminary data indicate that alterations in the cytoskeletal network do occur in cells obtained from older individuals. Alterations in actin state are also reported during malignant transformation of cells in culture, and in naturally occurring tumors. Taken together, the existing data seem to suggest that changes in the actin cytoskeletal network may be a part of the aging process as well as malignant transformation. Therefore, the study of the actin cytoskeletal network and its regulation has the potential to yield important information regarding cellular senescence and neoplastic transformation.     

A Prestressed Cable Network Model of the Adherent Cell Cytoskeleton

A prestressed cable network is used to model the deformability of the adherent cell actin cytoskeleton. The overall and microstructural model geometries and cable mechanical properties were assigned values based on observations from living cells and mechanical measurements on isolated actin filaments, respectively. The models were deformed to mimic cell poking (CP), magnetic twisting cytometry (MTC) and magnetic bead microrheometry (MBM) measurements on living adherent cells. The models qualitatively and quantitatively captured the fibroblast cell response to the deformation imposed by CP while exhibiting only some qualitative features of the cell response to MTC and MBM. The model for CP revealed that the tensed peripheral actin filaments provide the key resistance to indentation. The actin filament tension that provides mechanical integrity to the network was estimated at ~158 pN, and the nonlinear mechanical response during CP originates from filament kinematics. The MTC and MBM simulations revealed that the model is incomplete, however, these simulations show cable tension as a key determinant of the model response.