Actin polymerization controls the organization of WASH domains at the surface of endosomes

Sorting of cargoes in endosomes occurs through their selective enrichment into sorting platforms, where transport intermediates are generated. The WASH complex, which directly binds to lipids, activates the Arp2/3 complex and hence actin polymerization onto such sorting platforms. Here, we analyzed the role of actin polymerization in the physiology of endosomal domains containing WASH using quantitative image analysis. Actin depolymerization is known to enlarge endosomes. Using a novel colocalization method that is insensitive to the heterogeneity of size and shape of endosomes, we further show that preventing the generation of branched actin networks induces endosomal accumulation of the WASH complex. Moreover, we found that actin depolymerization induces a dramatic decrease in the recovery of endosomal WASH after photobleaching. This result suggests a built-in turnover, where the actin network, i.e. the product of the WASH complex, contributes to the dynamic exchange of the WASH complex by promoting its detachment from endosomes. Our experiments also provide evidence for a role of actin polymerization in the lateral compartmentalization of endosomes: several WASH domains exist at the surface of enlarged endosomes, however, the WASH domains coalesce upon actin depolymerization or Arp2/3 depletion. Branched actin networks are thus involved in the regulation of the size of WASH domains. The potential role of this regulation in membrane scission are discussed.     

The cellular regulation of vesicle exocytosis by Entamoeba histolytica

We studied the cellular regulation of vesicle exocytosis by Entamoeba histolytica utilizing release of endocytosed 125iodine (125I) labeled tyrosine conjugated dextran; 125I-dextran entered the acid pH vesicles of the amebae and was not degraded during these studies. Exocytosis was temperature dependent with 74%, 36%, 4%, and 0% of 125I-dextran released after 120 min at 37 degrees C, 31 degrees C, 25 degrees C, and 4 degrees C, respectively (P less than 0.01 for each). Exocytosis at 37 degrees C was inhibited by cytochalasin D (10 micrograms/ml), EDTA (10 mM), or the putative intracellular calcium antagonist TMB-8 (250 microM) (P less than 0.01 for each at greater than or equal to 60 min). Calcium ionophore A23187 (1 microM) enhanced exocytosis at 5 and 15 min (P less than 0.01). Elevation of vesicle pH with NH4Cl (10 mM) had no effect on release of 125I-dextran; phorbol myristate acetate (10(-6) M) increased exocytosis by 46% at 30 min (P less than 0.01). Centrifugation of amebae with target Chinese hamster ovary cells resulted in decreased 125I-dextran release into the cell supernatant after 30 and 60 min at 37 degrees C (by 40% and 42%, respectively, P less than 0.01); release of 125I-dextran returned to control values with addition of 1.0 g% galactose or GalNac but not with mannose or N-acetyl-D-glucosamine. Amebic phagocytosis of serum-exposed latex beads had no effect on release of dextran by amebae (n = 16). Exocytosis of acid pH vesicles by E. histolytica is temperature-, microfilament-, and calcium-dependent, and stimulated by phorbol esters.     

Actin polymerization and ATP hydrolysis

This paper surveys several aspects of the consequences of ATP hydrolysis associated with actin polymerization, and their physiological implications. ATP hydrolysis occurs on F-actin in two subsequent reactions, cleavage of ATP followed by the slower release of P_i. The latter reaction is linked to a conformation change of the actin subunit that causes a destabilization of the actin-actin interactions in the filament, i.e., a structural change of the filament. The nature of the nucleotide bound to terminal subunits therefore affects the dynamics of actin filaments. It is shown that this regulation is different at the two ends, terminal F-ADP-P_i subunits being present at steady state at the barbed end, while F-ADP-subunits are present at the pointed end. While cleavage of ATP on F-actin is irreversible, P_i release is reversible, which allows the regulation of filament dynamics by cellular P_i. The nature of the divalent metal ion — Ca²⁺ or Mg²⁺ — tightly bound to actin, in direct interaction with ATP, also affects the conformation of actin and the rate of ATP hydrolysis, therefore regulating actin dynamics. Finally, the rate of nucleotide exchange on G-actin is relatively slow, which allows the critical concentration to increase with the number of filaments in ATP, a property largely used by the cell via the action of severing proteins.     

Endosomal WASH and exocyst complexes control exocytosis of MT1-MMP at invadopodia

Remodeling of the extracellular matrix by carcinoma cells during metastatic dissemination requires formation of actin-based protrusions of the plasma membrane called invadopodia, where the trans-membrane type 1 matrix metalloproteinase (MT1-MMP) accumulates. Here, we describe an interaction between the exocyst complex and the endosomal Arp2/3 activator Wiskott-Aldrich syndrome protein and Scar homolog (WASH) on MT1-MMP–containing late endosomes in invasive breast carcinoma cells. We found that WASH and exocyst are required for matrix degradation by an exocytic mechanism that involves tubular connections between MT1-MMP–positive late endosomes and the plasma membrane in contact with the matrix. This ensures focal delivery of MT1-MMP and supports pericellular matrix degradation and tumor cell invasion into different pathologically relevant matrix environments. Our data suggest a general mechanism used by tumor cells to breach the basement membrane and for invasive migration through fibrous collagen-enriched tissues surrounding the tumor.     

Large dense-core vesicle exocytosis in pancreatic beta-cells monitored by capacitance measurements

This article discusses the currently used methodologies for monitoring exocytosis as changes in cell capacitance. Details are given on composition of solutions, experimental protocols, and how the observed responses can be interpreted physiologically. The concepts are illustrated by examples from our own work on insulin-releasing pancreatic beta-cells. Finally, we consider the feasibility of applying capacitance measurements to endocrine cells in intact pancreatic islets, where the cells are electrically coupled to each other.     

Imaging synaptic vesicle exocytosis and endocytosis with FM dyes

FM dyes have been used to label and then monitor synaptic vesicles, secretory granules and other endocytic structures in a variety of preparations. Here, we describe the general procedure for using FM dyes to study endosomal trafficking in general, and synaptic vesicle recycling in particular. The dye, dissolved in normal saline solution, is added to a chamber containing the preparation to be labeled. Stimulation evokes exocytosis, and compensatory endocytosis that follows traps FM dye inside the retrieved vesicles. The extracellular dye is then washed from the chamber, and labeled endocytic structures are examined with a fluorescence microscope. Fluorescence intensity provides a direct measure of the labeled vesicle number, a good measure of the amount of exocytosis. If the preparation is stimulated again, without dye in the chamber, dimming of the preparation provides a measure of exocytosis of labeled vesicles. With a synaptic preparation on hand, this protocol requires 1 day.     

Optical detection of synaptic vesicle exocytosis and endocytosis.

Recent advances in optical methods have catalyzed a detailed study of individual visualized synapses in several model systems. Quantal events at small central synapses, as well as single granule exocytosis in secretory cells, have been detected using quantitative fluorescence imaging. Sensitive detection of exocytosis and endocytosis at individual synapses has advanced our knowledge of synaptic vesicle trafficking.     

Actin filament polymerization regulates gliding motility by apicomplexan parasites

Host cell entry by Toxoplasma gondii depends critically on actin filaments in the parasite, yet paradoxically, its actin is almost exclusively monomeric. In contrast to the absence of stable filaments in conventional samples, rapid-freeze electron microscopy revealed that actin filaments were formed beneath the plasma membrane of gliding parasites. To investigate the role of actin filaments in motility, we treated parasites with the filament-stabilizing drug jasplakinolide (JAS) and monitored the distribution of actin in live and fixed cells using yellow fluorescent protein (YFP)-actin. JAS treatment caused YFP-actin to redistribute to the apical and posterior ends, where filaments formed a spiral pattern subtending the plasma membrane. Although previous studies have suggested that JAS induces rigor, videomicroscopy demonstrated that JAS treatment increased the rate of parasite gliding by approximately threefold, indicating that filaments are rate limiting for motility. However, JAS also frequently reversed the normal direction of motility, disrupting forward migration and cell entry. Consistent with this alteration, subcortical filaments in JAS-treated parasites occurred in tangled plaques as opposed to the straight, roughly parallel orientation observed in control cells. These studies reveal that precisely controlled polymerization of actin filaments imparts the correct timing, duration, and directionality of gliding motility in the Apicomplexa.     

The synaptic vesicle: cycle of exocytosis and endocytosis

Synaptic vesicles are clustered at the presynaptic terminal where they fuse and recycle in response to stimulation. Vesicles appear to be sorted into pools, but we do not yet understand how physiologically defined pools relate to morphological pools. The advent of dynamic imaging approaches has led to an appreciation of the regulation of vesicle mobility. Newly endocytosed vesicles are highly mobile but appear to become transiently trapped as they re-enter the recycling pool. Recent experiments indicate that endocytosis might have a constant rate, but limited capacity. How endocytosis is linked to exocytosis remains unclear, although calcium emerges as an important player.     

Synaptic vesicle exocytosis captured by quick freezing and correlated with quantal transmitter release

We describe the design and operation of a machine that freezes biological tissues by contact with a cold metal block, which incorporates a timing circuit that stimulates frog neuromuscular junctions in the last few milliseconds before thay are frozen. We show freeze-fracture replicas of nerve terminals frozen during transmitter discharge, which display synpatic vesicles caught in the act of exocytosis. We use 4-aminopyridine (4-AP) to increase the number of transmitter quanta discharged with each nerve impulse, and show that the number of exocytotic vesicles caught by quick-freezing increases commensurately, indicating that one vesicle undergoes exocytosis for each quantum that is discharged. We perform statistical analyses on the spatial distribution of synaptic vesicle discharge sites along the "active zones" that mark the secretory regions of these nerves, and show that individual vesicles fuse with the plasma membrane independent of one another, as expected from physiological demonstrations that quanta are discharged independently. Thus, the utility of quick-freezing as a technique to capture biological processes as evanescent as synaptic transmission has been established. An appendix describes a new capacitance method to measure freezing rates, which shows that the "temporal resolution" of our quick-freezing technique is 2 ms or better.     

Actin polymerization and synthesis in cultured neurones

The protein content of sympathetic neurones explanted from 10–11-day old chick embryos into culture medium containing nerve growth factor (NGF) increases steadily from about 100 to about 400 pg/cell in 7 days. Actin remains at close to 5% of the total protein during this period, but the proportion of unpolymerized actin falls. As measured by the inhibition of DNase I activity, rounded neurones without neurites contain 70 ± 7% of their total actin in monomeric form, whereas cells in mature, neurite-bearing cultures contain 39 ± 7%. When allowance is made for the increase in size of the neuronal cell bodies, the actin present in the neurites (‘axons’) alone is found to be almost entirely in filamentous form.Cultures exposed to radioactive leucine rapidly incorporate radioactivity into both sedimentable and non-sedimentable forms of actin. Actin-specific activities in the two fractions—estimated after isolation of the actin on small DNase I—Sepharose affinity columns—are similar after labelling for less than 1 h. Direct incorporation of newly-synthesized actin into filaments is suggested from these results.Pulse-chase experiments show that non-sedimentable protein in cultured sympathetic neurones turns over more rapidly than sedimentable protein. However, this is not true for actin, which shows a similar specific activity in sedimentable and non-sedimentable forms—even after 6 days of cold chase. This anomalous behaviour is simply explained by an exchange of actin molecules between filamentous and non-filamentous forms. Control experiments indicate that exchange does not occur to this degree during preparation of subcellular fractions. It is consequently attributed to exchange processes in the living cell.     

Dynamics of membranes driven by actin polymerization

A motile cell, when stimulated, shows a dramatic increase in the activity of its membrane, manifested by the appearance of dynamic membrane structures such as lamellipodia, filopodia, and membrane ruffles. The external stimulus turns on membrane bound activators, like Cdc42 and PIP2, which cause increased branching and polymerization of the actin cytoskeleton in their vicinity leading to a local protrusive force on the membrane. The emergence of the complex membrane structures is a result of the coupling between the dynamics of the membrane, the activators, and the protrusive forces. We present a simple model that treats the dynamics of a membrane under the action of actin polymerization forces that depend on the local density of freely diffusing activators on the membrane. We show that, depending on the spontaneous membrane curvature associated with the activators, the resulting membrane motion can be wavelike, corresponding to membrane ruffling and actin waves, or unstable, indicating the tendency of filopodia to form. Our model also quantitatively explains a variety of related experimental observations and makes several testable predictions.     

Cell motility driven by actin polymerization.

Certain kinds of cellular movements are apparently driven by actin polymerization. Examples include the lamellipodia of spreading and migrating embryonic cells, and the bacterium Listeria monocytogenes, that propels itself through its host's cytoplasm by constructing behind it a polymerized tail of cross-linked actin filaments. Peskin et al. (1993) formulated a model to explain how a polymerizing filament could rectify the Brownian motion of an object so as to produce unidirectional force (Peskin, C., G. Odell, and G. Oster. 1993. Cellular motions and thermal fluctuations: the Brownian ratchet. Biophys. J. 65:316-324). Their "Brownian ratchet" model assumed that the filament was stiff and that thermal fluctuations affected only the "load," i.e., the object being pushed. However, under many conditions of biological interest, the thermal fluctuations of the load are insufficient to produce the observed motions. Here we shall show that the thermal motions of the polymerizing filaments can produce a directed force. This "elastic Brownian ratchet" can explain quantitatively the propulsion of Listeria and the protrusive mechanics of lamellipodia. The model also explains how the polymerization process nucleates the orthogonal structure of the actin network in lamellipodia.     

Actin polymerization overshoots and ATP hydrolysis as assayed by pyrene fluorescence

We investigate via stochastic simulation the overshoots observed in the fluorescence intensity of pyrene-labeled actin during rapid polymerization. We show that previous assumptions about pyrene intensity that ignore the intensity differences between subunits in different ATP hydrolysis states are not consistent with experimental data. This strong sensitivity of intensity to hydrolysis state implies that a measured pyrene intensity curve does not immediately reveal the true polymerization kinetics. We show that there is an optimal range of hydrolysis and phosphate release rate combinations simultaneously consistent with measured polymerization data from previously published severing and Arp2/3 complex-induced branching experiments. Within this range, we find that the pyrene intensity curves are described very accurately by the following average relative intensity coefficients: 0.37 for F-ATP actin; 0.55 for F-ADP + P_i actin; and 0.75 for F-ADP actin. Finally, we present an analytic formula, which properly accounts for the sensitivity of the pyrene assay to hydrolysis state, for estimation of the concentration of free barbed ends from pyrene intensity curves.     

囊泡胞吐机制及与其相关的神经元可塑性

突触前囊泡释放神经递质经历了磷酸化synapsin I使囊泡脱离细胞骨架,Rab3A导引囊泡进入突触前膜激活区,Rab3A与RIMl结合介导的囊泡锚定,Munc-13—1引燃SNARE中心复合体装配,最后Ca^2 结合到Ca^2 传感器synaptotagmin触发囊泡融合,融合后的囊泡通过SNAP和NSF的作用,使SNARE复合体解体后经内吞机制形成新的囊泡参与再循环。研究表明,参与囊泡融合的分子元件在神经元可塑性中发挥重要作用。     

Complexin synchronizes primed vesicle exocytosis and regulates fusion pore dynamics

ComplexinII (CpxII) and SynaptotagminI (SytI) have been implicated in regulating the function of SNARE proteins in exocytosis, but their precise mode of action and potential interplay have remained unknown. In this paper, we show that CpxII increases Ca²⁺-triggered vesicle exocytosis and accelerates its secretory rates, providing two independent, but synergistic, functions to enhance synchronous secretion. Specifically, we demonstrate that the C-terminal domain of CpxII increases the pool of primed vesicles by hindering premature exocytosis at submicromolar Ca²⁺ concentrations, whereas the N-terminal domain shortens the secretory delay and accelerates the kinetics of Ca²⁺-triggered exocytosis by increasing the Ca²⁺ affinity of synchronous secretion. With its C terminus, CpxII attenuates fluctuations of the early fusion pore and slows its expansion but is functionally antagonized by SytI, enabling rapid transmitter discharge from single vesicles. Thus, our results illustrate how key features of CpxII, SytI, and their interplay transform the constitutively active SNARE-mediated fusion mechanism into a highly synchronized, Ca²⁺-triggered release apparatus.     

SNARE complex oligomerization by synaphin/complexin is essential for synaptic vesicle exocytosis

Synaphin/complexin is a cytosolic protein that preferentially binds to syntaxin within the SNARE complex. We find that synaphin promotes SNAREs to form precomplexes that oligomerize into higher order structures. A peptide from the central, syntaxin binding domain of synaphin competitively inhibits these two proteins from interacting and prevents SNARE complexes from oligomerizing. Injection of this peptide into squid giant presynaptic terminals inhibited neurotransmitter release at a late prefusion step of synaptic vesicle exocytosis. We propose that oligomerization of SNARE complexes into a higher order structure creates a SNARE scaffold for efficient, regulated fusion of synaptic vesicles.     

Actin polymerization and pseudopod extension during amoeboid chemotaxis

Amoebae of the cellular slime mold Dictyostelium discoideum are an excellent model system for the study of amoeboid chemotaxis. These cells can be studied as a homogeneous population whose response to chemotactic stimulation is sufficiently synchronous to permit the correlation of the changes in cell shape and biochemical events during chemotaxis. Having demonstrated this synchrony of response, we show that actin polymerization occurs in two stages during stimulation with chemoattractants. The assembly of F-actin that peaks between 40 and 60 sec after the onset of stimulation is temporally correlated with the growth of new pseudopods. F-actin, which is assembled by 60 sec after stimulation begins, is localized in the new pseudopods that are extended at this time. Both stages of actin polymerization during chemotactic stimulation involve polymerization at the barbed ends of actin filaments based on the cytochalasin sensitivity of this response. We present a hypothesis in which actin polymerization is one of the major driving forces for pseudopod extension during chemotaxis. The predictions of this model, that localized regulation of actin nucleation activity and actin filament cross-linking must occur, are discussed in the context of current models for signal transduction and of recent information regarding the types of actin-binding proteins that are present in the cell cortex.     

Actin polymerization induced by chemotactic peptide and concanavalin A in rat neutrophils

Changes in the state of actin in rat neutrophils were studied after chemotactic peptide and concanavalin A stimulation by using the DNase I inhibition assay. Actin polymerization occurred within seconds after stimulation with F-Met-Leu-Phe and concanavalin A. Pretreatment of cells with cytochalasin D prevented chemotactic peptide-induced actin polymerization. The addition of F-Met-Leu-Phe to lysed cells did not produce any change in actin state. These data offer strong evidence for receptor-induced actin polymerization and support the models implicating actin microfilament formation as a crucial event in cell activation. The observations on platelets, lymphocytes, neutrophils, and islets of Langerhans from different species suggest that actin polymerization might be a universal intracellular event accompanying cell surface receptor perturbation in eukaryotic cells.