Population Of Filaments Research Articles

Characterization of Osmotically Induced Filaments of Salmonella enterica

Salmonella enterica forms aseptate filaments with multiple nucleoids when cultured in hyperosmotic conditions. These osmotic-induced filaments are viable and form single colonies on agar plates even though they contain multiple genomes and have the potential to divide into multiple daughter cells. Introducing filaments that are formed during osmotic stress into culture conditions without additional humectants results in the formation of septa and their division into individual cells, which could present challenges to retrospective analyses of infectious dose and risk assessments. We sought to characterize the underlying mechanisms of osmotic-induced filament formation. The concentration of proteins and chromosomal DNA in filaments and control cells was similar when standardized by biomass. Furthermore, penicillin-binding proteins in the membrane of salmonellae were active in vitro. The activity of penicillin-binding protein 2 was greater in filaments than in control cells, suggesting that it may have a role in osmotic-induced filament formation. Filaments contained more ATP than did control cells in standardized cell suspensions, though the levels of two F(0)F(1)-ATP synthase subunits were reduced. Furthermore, filaments could septate and divide within 8 h in 0.2 × Luria-Bertani broth at 23°C, while nonfilamentous control cells did not replicate. Based upon the ability of filaments to septate and divide in this diluted broth, a method was developed to enumerate by plate count the number of individual, viable cells within a population of filaments. This method could aid in retrospective analyses of infectious dose of filamented salmonellae.

Abstract 5172: The role of vimentin in FAK-mediated cell adhesion and metastasis

Abstract Lung cancer is the leading cause of cancer death in the United States. Specifcally, non-small cell lung cancer (NSCLC) is responsible for about 80% of lung cancer deaths and has a 5-year survival rate of only 15-25%. This poor survival statistic results from diagnosis at advanced stages of the disease when metastasis of the primary tumor has already occurred; therefore, identifying the molecular mechanisms guiding metastasis is crucial for advances in treatment. The intermediate filament vimentin is overexpressed in metastatic cancers and its expression correlates with increased invasive capacity and reduced patient survival. A population of vimentin filaments localizes to focal adhesions, which attach to the extracellular matrix and are required for cell motility. Focal adhesions are composed of a complex of proteins, including focal adhesion kinase (FAK), which is overexpressed in NSCLC. Since evidence suggests that both FAK and vimentin co-localize at focal adhesions, we hypothesize that vimentin acts through FAK to regulate cell adhesion and thereby cell migration. Our results show that vimentin depletion leads to decreased cell adhesion and migration, with decreased FAK activation and expression. We show that vimentin filaments directly enter total FAK and pFAK- Y397 sites at the cellular leading edge of motile lung cancer cells. Using immunoprecipitation, we show vimentin interacts with several members of the focal adhesion complex, suggesting that vimentin can regulate focal adhesion activity. To address the mechanism by which this may occur, we hypothesize that the phosphorylation state of vimentin, which is important for cell migration, is crucial for proper functioning at focal adhesions. This is the focus of our future work with the goal of identifying potential new therapeutic targets to block metastasis and improve lung cancer patients’ prognosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5172. doi:1538-7445.AM2012-5172

Abstract 1827: Developing chemotherapeutics which selectively disable the actin cytoskeleton of tumor cells

Abstract The actin cytoskeleton is an important regulator of a variety of cellular functions making it an ideal chemotherapeutic target. Despite this promise no anti-actin compounds are used in current chemotherapy, primarily due to the inability of existing anti-actin agents to discriminate between the actin cytoskeleton of tumor cells and that of the muscle sarcomere. We have previously shown that tropomyosin, an integral component of the actin cytoskeleton, defines functionally distinct actin filament populations. We have designed a new class of compounds which target a specific cancer associated tropomyosin isoform, Tm5NM1. Anti-tropomyosin (Tm) compounds were selected based on ability to target the actin cytoskeleton and efficacy against a panel of neuroblastoma and melanoma cell lines. The lead compound, TR100, was shown to be effective against a panel of tumor cell lines (average EC50 3µM). Preliminary studies demonstrate that TR100 induces cell death via the intrinsic (mitochondrial) apoptotic pathway and the mechanism appears to be independent of the pro apoptotic factors BAX and BAK. In 3D melanoma spheroid models, which more accurately mimic the tumor microenvironment, TR100 inhibited cell growth and motility. This translated to a reduction in tumor cell growth in neuroblastoma and melanoma xenograft models. Analysis of drug treated animals showed no evidence of cardiac damage as measured by blood Troponin I levels and the intraventricular septum thickness of isolated hearts. These results demonstrate the possibility of targeting distinct actin filament populations based on tropomyosin composition. Next generation anti-Tm compounds with improved efficacy and specificity have now been developed. Preliminary data demonstrate that these compounds exhibit increased selectivity for transformed cells. Taken together, our findings suggest that the anti-Tm compounds show a significant improvement in the therapeutic window compared to existing anti-actin agents. This novel approach and the development of the new class of anti-Tm compounds may be the key for disabling a long sought after target, the actin cytoskeleton, and may lead to a new class of chemotherapeutics active against a broad range of cancer types. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1827. doi:1538-7445.AM2012-1827

Self-Assembly of Silk-Collagen-like Triblock Copolymers Resembles a Supramolecular Living Polymerization

We produced several pH-responsive silk-collagen-like triblocks, one acidic and two alkaline. At pH values where the silk-like block is uncharged the triblocks self-assemble into filaments. The pH-induced self-assembly was examined by atomic force microscopy, light scattering, and circular dichroism. The populations of filaments were found to be very monodisperse, indicating that the filaments start to grow from already present nuclei in the sample. The growth then follows pseudo-first-order kinetics for all examined triblocks. When normalized to the initial concentration, the growth curves of each type of triblock overlap, showing that the self-assembly is a generic process for silk-collagen-silk triblocks, regardless of the nature of their chargeable groups. The elongation speed of the filaments is slow, due to the presence of repulsive collagen-like blocks and the limited number of possibilities for an approaching triblock to successfully attach to a growing end. The formation of filaments is fully reversible. Already present filaments can start growing again by addition of new triblocks. The structure of all filaments is very rich in β-turns, leading to β-rolls. The triblocks attain this structure only when attaching to a growing filament.

Abstract A178: Developing chemotherapeutics which selectively disable the actin cytoskeleton of tumor cells.

Abstract Background: The actin cytoskeleton is an important regulator of a variety of cellular functions including cell motility, adhesion, and proliferation, making it an ideal chemotherapeutic target. Despite this promise there are still no anti-actin compounds used in current chemotherapy, primarily due to the inability of existing anti-actin agents to discriminate between the actin cytoskeleton of tumor cells and the actin filaments of the muscle sarcomere. We have previously shown that tropomyosin, an integral component of the actin cytoskeleton, defines functionally distinct populations of actin filaments. We have identified a specific tropomyosin isoform common to all tumor cells tested to date which regulates cell proliferation and have designed a new class of compounds to target this filament population. Summary of Results: Anti-tropomyosin (Tm) compounds were selected based their ability to target the actin cytoskeleton and their efficacy against a panel of neuroblastoma and melanoma cell lines. The lead compound, TR100, was shown to be effective against a panel of tumor cell lines with an average EC50 of 3μM. When tested in 3D melanoma spheroid models, which more accurately mimic the tumor microenvironment, TR100 inhibited melanoma cell growth and motility. This effect translated to a reduction in tumor cell growth in vivo in both neuroblastoma and melanoma xenograft models. In vitro data using isolated rat cardiomyocytes demonstrated that TR100 had minimal impact on contractile function. In vivo data from the drug treated animals also showed no evidence of cardiac damage as measured by blood Troponin I levels and no changes in the intraventricular septum thickness of isolated hearts. These results demonstrate that it is possible to target distinct actin filament populations based on the tropomyosin composition. Next generation anti-Tm compounds with improved efficacy and specificity have now been developed. Preliminary data demonstrate that these compounds exhibit increased selectivity for transformed cells. Conclusions: Taken together, our findings suggest that the anti-Tm compounds show a significant improvement in the therapeutic window compared to existing anti-actin agents. This novel approach and the development of the new class of anti-Tm compounds may be the key for disabling a long sought after target, the actin cytoskeleton, and may lead to a new class of chemotherapeutics active against a broad range of cancer types. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A178.

Microfluidics pushes forward microscopy analysis of actin dynamics

Actin filaments, an essential part of the cytoskeleton, drive various cell processes, during which they elongate, disassemble and form different architectures. Over the past 30 years, the study of actin dynamics has relied mainly on bulk solution measurements, which revealed the kinetics and thermodynamics of actin self-assembly at barbed and pointed ends, its control by ATP hydrolysis and its regulation by proteins binding either monomeric actin or filament ends and sides. These measurements provide quantitative information on the averaged behavior of a homogeneous population of filaments. They have been complemented by light microscopy observations of stabilized individual filaments, providing information inaccessible using averaging methods, such as mechanical properties or length distributions. In the past ten years, the improvement of light microscopy techniques has allowed biophysicists to monitor the dynamics of individual actin filaments, thus giving access to the length fluctuations of filaments or the mechanism of processive assembly by formins. Recently, in order to solve some of the problems linked to these observations, such as the need to immobilize filaments on a coverslip, we have used microfluidics as a tool to improve the observation, manipulation and analysis of individual actin filaments. This microfluidic method allowed us to rapidly switch filaments from polymerizing to depolymerizing conditions, and derive the molecular mechanism of ATP hydrolysis on a single filament from the kinetic analysis of its nucleotide-dependent disassembly rate. Here, we discuss how this work sets the basis for future experiments on actin dynamics, and briefly outline promising developments of this technique.

TPM3 and TPM4 gene products segregate to the postsynaptic region of central nervous system synapses

Synaptic function in the central nervous system (CNS) is highly dependent on a dynamic actin cytoskeleton in both the pre- and the postsynaptic compartment. Remodelling of the actin cytoskeleton is controlled by tropomyosins, a family of actin-associated proteins which define distinct actin filament populations. Here we show that TPM3 and TPM4 gene products localize to the postsynaptic region in mouse hippocampal neurons. Furthermore our data confirm previous findings of isoform segregation to the pre- and postsynaptic compartments at CNS synapses. These data provide fundamental insights in the formation of functionally distinct actin filament populations at the pre- and post-synapse.

Septin GTPases spatially guide microtubule organization and plus end dynamics in polarizing epithelia

Establishment of epithelial polarity requires the reorganization of the microtubule (MT) cytoskeleton from a radial array into a network positioned along the apicobasal axis of the cell. Little is known about the mechanisms that spatially guide the remodeling of MTs during epithelial polarization. Septins are filamentous guanine triphosphatases (GTPases) that associate with MTs, but the function of septins in MT organization and dynamics is poorly understood. In this paper, we show that in polarizing epithelia, septins guide the directionality of MT plus end movement by suppressing MT catastrophe. By enabling persistent MT growth, two spatially distinct populations of septins, perinuclear and peripheral filaments, steer the growth and capture of MT plus ends. This navigation mechanism is essential for the maintenance of perinuclear MT bundles and for the orientation of peripheral MTs as well as for the apicobasal positioning of MTs. Our results suggest that septins provide the directional guidance cues necessary for polarizing the epithelial MT network.

Building Distinct Actin Filament Networks in a Common Cytoplasm

Eukaryotic cells generate a diversity of actin filament networks in a common cytoplasm to optimally perform functions such as cell motility, cell adhesion, endocytosis and cytokinesis. Each of these networks maintains precise mechanical and dynamic properties by autonomously controlling the composition of its interacting proteins and spatial organization of its actin filaments. In this review, we discuss the chemical and physical mechanisms that target distinct sets of actin-binding proteins to distinct actin filament populations after nucleation, resulting in the assembly of actin filament networks that are optimized for specific functions.

Planktothrix populations in subalpine lakes: selection for strains with strong gas vesicles as a function of lake depth, morphometry and circulation

Summary 1. The genus Planktothrix (Cyanobacteria) usually produces concentrated populations of filaments in the summer metalimnion of thermally stratifying lakes. This has been associated with the action of gas vesicles, cellular structures providing positive buoyancy. At the end of the summer, filaments are carried by convective mixing deeper into the water column where some gas vesicles collapse as a result of high hydrostatic pressure. They then lose their buoyancy, sink and are lost from the euphotic zone. 2. The resistance of gas vesicles to hydrostatic pressures is critical for the survival of Planktothrix in deep lakes. However, comparative observations on populations from lakes of a range of depths and hydrodynamic regimes are still needed to examine the relationships between the adaptive trait (i.e. the ‘critical’ pressure at which each gas vesicle collapses) with the environmental factor (i.e. the maximum hydrostatic pressure). 3. To explore the adaptation of Planktothrix populations to the depth of winter circulation in different systems, we collected 276 strains of P. cf. rubescens from eight lakes (zmax = 24–410 m) in Northern Italy during summer 2009 and we analysed the multicopy gene gvpC coding for a protein that crucially influences the critical pressure. 4. The strains analysed clustered into two main groups having gas vesicles with a mean critical pressure of 1.1 and 0.9 MPa, respectively. The proportion of the stronger strains was generally positively related to lake depth, although the overall pattern was complicated by individual lake morphology and hydrology. The relative frequency of stronger filaments was (i) greatest in deep basins with concave slopes and (ii) least in one deep, but permanently stratified lake. 5. The simultaneous presence of ‘weaker’ and ‘stronger’ filaments could allow for a rapid adaptive response to changes in hydrostatic pressures, related to changes in the amplitude of vertical circulation characterising deep lakes.

Tropomyosin isoform 3 promotes the formation of filopodia by regulating the recruitment of actin-binding proteins to actin filaments

Tropomyosins are believed to function in part by stabilizing actin filaments. However, accumulating evidence suggests that fundamental differences in function exist between tropomyosin isoforms, which contributes to the formation of functionally distinct filament populations. We investigated the functions of the high-molecular-weight isoform Tm3 and examined the molecular properties of Tm3-containing actin filament populations. Overexpression of the Tm3 isoform specifically induced the formation of filopodia and changes in actin solubility. We observed alterations in actin-binding protein recruitment to filaments, co-incident with changes in expression levels, which can account for this functional outcome. Tm3-associated filaments recruit active actin depolymerizing factor and are bundled into filopodia by fascin, which is both up-regulated and preferentially associated with Tm3-containing filaments in the Tm3 overexpressing cells. This study provides further insight into the isoform-specific roles of different tropomyosin isoforms. We conclude that variation in the tropomyosin isoform composition of microfilaments provides a mechanism to generate functionally distinct filament populations.

Sinking rates of phytoplankton filaments orientated at different angles: theory and physical model

Measurements of phytoplankton sinking rates are usually performed on samples of randomly oriented cells or colonies and yet the theoretical analyses of results are usually based on formulae restricted to objects oriented either vertically or horizontally. Formulae first published in 1965 but unknown in the phytoplankton literature can be used to calculate sinking rates of prolate ellipsoids and cylinders, shapes commonly encountered in phytoplankton, sinking at a range of angles between vertical and horizontal. The formulae predict a curvilinear decrease in speed from the maximum when oriented at 0° to the vertical, and a horizontal component in the trajectories of cylindrical particles oriented at angles of >0° and <90°, with filaments with an axial ratio of 100 drifting sideways at up to 25% of the speed of vertical drift. All of these predictions were verified with a physical model of graphite cylinders (pencil leads) sinking in an oil-filled sedimentation column, after corrections for column wall effects were applied. The mean vertical sinking rate of a disperse, randomly oriented population of filaments was also calculated, from the probability of orientation at each angle, and the theoretical speed at that angle.

The Molecular Adaptations of Myosin X for Bundled Actin Filament Tracks

To properly self-organize, cells must be able to direct cargo to specific locations. Although many molecular motors are known to drive cargo transport, the address system that these motors use to move to the proper destination is poorly understood. Recently, we showed that myosin X has a preference for bundled actin filaments. This preference for bundles allows myosin X to identify filopodia, a limited population of actin filaments within the cell. To clarify the bundle selection mechanism, we performed single-molecule mechanical measurements to determine the stepping pattern on bundles. Our observed ∼18 nm stepsize is consistent with a filament-straddling mechanism, where each head of myosin X binds to a unique filament in the bundle. A dissection of the domains required for bundle selection reveals a surprising role for the myosin X tail, a region which is likely far from the bundle itself. We find that targeted insertion of a glycine-rich flexible linker within the tail abolishes bundle selectivity, suggesting that the tail adopts a rigid structure that is essential for identifying bundles.

Glimpses on microtubule accessory proteins

Microtubules allow cells to regulate their architecture, movement, and division in a very flexible manner and in concert with the signals and challenges they perceive from the environment. Surprisingly, this versatile tool basically consists of heterodimers of two very similar proteins that are even fairly conserved over evolution. What qualifies these organelles to fulfill so many tasks and functions? Due to the molecular conservation of their building elements, the tubulins, it is generally believed that the functional specificity of microtubules is mediated by associated proteins. In fact, there exists a wealth of such accessory proteins that, during evolution, have diversified considerably. Two contributions in the present issue address functional and evolutionary aspects of such microtubule accessory proteins. The maize mutant brick1 is affected in epidermal morphogenesis as result of altered WAVE/ARP2/3 function. In an attempt to understand the role of the BRICK1 protein in cell division, Panteris et al., in the current issue, follow mitotic actin arrays and observe that, in contrast to the wild type, actin accumulates in the preprophase band of dividing protodermal cells of the mutant. In addition, actin accumulates in mutant spindles after treatment with taxol. Interestingly, this does not affect neither spindle structure nor division activity. This work contributes to the discussion on the role of actomyosin in mitosis (see editorial of the last issue). Based on evolutionary considerations, it is argued that, most likely, cells adopted pre-existing systems of interphase motility to generate the forces required for chromosomal translocation. In fact, there is evidence from insect cells for a role of actomyosin in mitosis. In plant cells, the colocalization of the so-called phragmosome with the preprophase band has been known for a long time. However, the link between microtubules and actin disappears during establishment of the division spindle, but is re-established during late anaphase, when the formation of the phragmoplast is initiated. This shows that, in plant cells as well, mitotic microtubule arrays are acting in concerted action with actin filaments and that BRICK1 is a key regulator for this interaction. On the other hand, the supernumerous actin filaments in preprophase band and spindle seem to have little effect neither for function and organization of mitotic microtubules nor cell division. This, at first glance, would contradict the spindle matrix model. However, it would be naive to assume a homogenous population of actin filaments. The effect of altered BRICK1 function in the mutant is mainly expected for filaments endowed with high nucleation dynamics, whereas functions driven by stable actin filaments (as those participating in myosin-driven movement) should remain largely untouched. The findings reported by Panteris et al. indicate, therefore, that we might be able to dissect the cross-talk between microtubules and actin filaments with respect to functionally and dynamically different subpopulations of actin. The evolutionary origin of microtubule organization has been associated with the basal body. This organizer of the flagellar apparatus is designed to ensure multiple functions such as locomotion, sensory perception, and cell division. In many flagellate species, these basal bodies are tethered by a relatively stiff, noncontractile structure to the plasma membrane of the cell. These socalled striated roots accompany microtubules in green algae and are composed of striated fiber assemblin, an acidic protein structurally related to β-giardin. In addition Protoplasma (2009) 236:1–2 DOI 10.1007/s00709-009-0054-7

Vimentin Intermediate Filament Formation: In Vitro Measurement and Mathematical Modeling of the Filament Length Distribution during Assembly

The salt-induced in vitro assembly of cytoplasmic intermediate filament (IF) proteins such as vimentin is characterized by a very rapid lateral association of soluble tetrameric subunits into 60-nm-long full-width "unit-length" filaments (ULFs). We have demonstrated for this prototype IF protein that filament elongation occurs by the longitudinal annealing of ULFs into short IFs. These IFs further longitudinally anneal and thus constitute a progressively elongating filament population that over time yields filaments of several microm in length. Previously, we provided a mathematical model for the kinetics of the assembly process based on the average length distribution of filaments as determined by time-lapse electron and atomic force microscopy. Thereby, we were able to substantiate the concept that end-to-end-annealing of both ULFs and short filaments is obligatory for the formation of long IFs (Kirmse, R.; Portet, S.; Mücke, N. Aebi, U.; Herrmann, H.; Langowski, J. J. Biol. Chem. 2007, 282, 18563-18572). As the next step in understanding the mechanics of IF formation, we have expanded our mathematical model to describe the quantitative aspects of IF assembly by taking into account geometry constraints as well as the diffusion properties of rodlike linear aggregates. Thereby, we have developed a robust model for the time-dependent filament length distribution of IFs under standard conditions.

Direct visualisation and kinetic analysis of normal and nemaline myopathy actin polymerisation using total internal reflection microscopy

Actin filaments were formed by elongation of pre-formed nuclei (short crosslinked actin-HMM complexes) that were attached to a microscope cover glass. By using TIRF illumination we could see actin filaments at high contrast despite the presence of 150 nM TRITC-phalloidin in the solution. Actin filaments showed rapid bending and translational movements due to Brownian motion but the presence of the methylcellulose polymer network constrained lateral movement away from the surface. Both the length and the number of filaments increased with time. Some filaments did not change length at all and some filaments joined up end-to-end (annealing). We did not see any decrease in filament length or filament breakage. For quantitative analysis of polymerisation time course we measured the contour length of all the filaments in a frame at a series of time points and also tracked the length of individual filaments over time. Elongation rate was the same measured by both methods (0.23 microm/min at 0.1 microM actin) and was up to 10 times faster than previously published measurements. The annealed filament population reached 30% of the total after 40 min. Polymerisation rate increased linearly with actin concentration. K(on) was 2.07 microm min(-1) microM(-1) (equivalent to 34.5 monomers s(-1) microM(-1)) and critical concentration was less than 20 nM. This technique was used to study polymerisation of a mutant actin (D286G) from a transgenic mouse model. D286G actin elongated at a 40% lower rate than non-transgenic actin.

Actin filament dynamics are dominated by rapid growth and severing activity in the Arabidopsis cortical array

Metazoan cells harness the power of actin dynamics to create cytoskeletal arrays that stimulate protrusions and drive intracellular organelle movements. In plant cells, the actin cytoskeleton is understood to participate in cell elongation; however, a detailed description and molecular mechanism(s) underpinning filament nucleation, growth, and turnover are lacking. Here, we use variable-angle epifluorescence microscopy (VAEM) to examine the organization and dynamics of the cortical cytoskeleton in growing and nongrowing epidermal cells. One population of filaments in the cortical array, which most likely represent single actin filaments, is randomly oriented and highly dynamic. These filaments grow at rates of 1.7 µm/s, but are generally short-lived. Instead of depolymerization at their ends, actin filaments are disassembled by severing activity. Remodeling of the cortical actin array also features filament buckling and straightening events. These observations indicate a mechanism inconsistent with treadmilling. Instead, cortical actin filament dynamics resemble the stochastic dynamics of an in vitro biomimetic system for actin assembly.

LIM Kinase 1 and Cofilin Regulate Actin Filament Population Required for Dynamin-dependent Apical Carrier Fission from theTrans-Golgi Network

The functions of the actin cytoskeleton in post-Golgi trafficking are still poorly understood. Here, we report the role of LIM Kinase 1 (LIMK1) and its substrate cofilin in the trafficking of apical and basolateral proteins in Madin-Darby canine kidney cells. Our data indicate that LIMK1 and cofilin organize a specialized population of actin filaments at the Golgi complex that is selectively required for the emergence of an apical cargo route to the plasma membrane (PM). Quantitative pulse-chase live imaging experiments showed that overexpression of kinase-dead LIMK1 (LIMK1-KD), or of LIMK1 small interfering RNA, or of an activated cofilin mutant (cofilin S3A), selectively slowed down the exit from the trans-Golgi network (TGN) of the apical PM marker p75-green fluorescent protein (GFP) but did not interfere with the apical PM marker glycosyl phosphatidylinositol-YFP or the basolateral PM marker neural cell adhesion molecule-GFP. High-resolution live imaging experiments of carrier formation and release by the TGN and analysis of peri-Golgi actin dynamics using photoactivatable GFP suggest a scenario in which TGN-localized LIMK1-cofilin regulate a population of actin filaments required for dynamin-syndapin-cortactin-dependent generation and/or fission of precursors to p75 transporters.

Actin Dynamics Is Essential for Myosin-Based Transport of Membrane Organelles

Actin filaments that serve as "rails" for the myosin-based transport of membrane organelles [1-4] continuously turn over by concurrent growth and shortening at the opposite ends [5]. Although it is known that dynamics of actin filaments is essential for many of the actin cytoskeleton functions, the role of such dynamics in myosin-mediated organelle transport was never studied before. Here, we addressed the role of turnover of actin filaments in the myosin-based transport of membrane organelles by treating cells with the drugs that suppress actin-filament dynamics and found that such a suppression significantly inhibited organelle transport along the actin filaments without inhibiting their intracellular distribution or the activity of the myosin motors. We conclude that dynamics of actin filaments is essential for myosin-based transport of membrane organelles and suggest a previously unknown role of actin-filament dynamics in providing the "rails" for continuous organelle movement resulting in the increased distances traveled by membrane organelles along the actin filaments.

Performance of a Population of Independent Filaments in Lamellipodial Protrusion

Actin polymerization is responsible for moving a wide variety of loads, from the protrusion of membrane-bound filopodia and lamellipodia of immune, cancer, and other motile cells, to the propulsion of some intracellular pathogens. A universal explanation of the forces and velocities generated by these systems has been hampered by a lack of understanding in how a population of independent filaments pushes these loads. Protrusion of a lamellipodium by the very filaments supporting the membrane load is thought to operate by the Brownian ratchet mechanism, with overall organization governed by the dendritic-nucleation/array-treadmilling model. We have incorporated these two models into a two-dimensional, stochastic computer model of lamellipodial protrusion, and studied how force and velocity generation varied under different assumptions. Performance is very sensitive to the extent to which the work of protrusion is shared among individual polymerization events within the filament population. Three identified mechanisms promote this “work-sharing”: 1), Most systems, including lamellipodia, utilize a self-organizing distribution of filament-load distances which serves to decrease the effective size of a monomer and dramatically improve performance. 2), A flexible membrane allows for consistent performance over wide leading edges. 3), Finally, very flexible filaments are capable of sharing work very uniformly, and therefore, of near-perfect theoretical performance. Transient tethering to the lamellipodial membrane limits their efficacy, however, and mandates a minimum filament stiffness. Overall, we estimate lamellipodia to operate with 40-nm bending-length filaments and low characteristic tether forces. Modeled lamellipodia exhibit sigmoidal force-velocity relationships and share the work of protrusion only moderately well among filaments, performing at approximately one-half of theoretical force and velocity maximums. At this level of work-sharing, the natural monomer size is optimal for generating velocity.