The Chlamydomonas Mating Type Plus Fertilization Tubule, a Prototypic Cell Fusion Organelle: Isolation, Characterization, and In Vitro Adhesion to Mating Type Minus Gametes

In the biflagellated alga Chlamydomonas, adhesion and fusion of the plasma membranes of gametes during fertilization occurs via an actin-filled, microvillus-like cell protrusion. Formation of this ~3-μm-long fusion organelle, the Chlamydomonas fertilization tubule, is induced in mating type plus (mt+) gametes during flagellar adhesion with mating type minus (mt−) gametes. Subsequent adhesion between the tip of the mt+ fertilization tubule and the apex of a mating structure on mt− gametes is followed rapidly by fusion of the plasma membranes and zygote formation. In this report, we describe the isolation and characterization of fertilization tubules from mt+ gametes activated for cell fusion. Fertilization tubules were detached by homogenization of activated mt+ gametes in an EGTA-containing buffer and purified by differential centrifugation followed by fractionation on sucrose and Percoll gradients. As determined by fluorescence microscopy of samples stained with a fluorescent probe for filamentous actin, the method yielded 2–3 × 10⁶ fertilization tubules/μg protein, representing up to a 360-fold enrichment of these organelles. Examination by negative stain electron microscopy demonstrated that the purified fertilization tubules were morphologically indistinguishable from fertilization tubules on intact, activated mt+ gametes, retaining both the extracellular fringe and the internal array of actin filaments. Several proteins, including actin as well as two surface proteins identified by biotinylation studies, copurified with the fertilization tubules. Most importantly, the isolated mt+ fertilization tubules bound to the apical ends of activated mt− gametes between the two flagella, the site of the mt− mating structure; a single fertilization tubule bound per cell, binding was specific for gametes, and fertilization tubules isolated from trypsin-treated, activated mt+ gametes did not bind to activated mt− gametes.     

Lipid raft–dependent plasma membrane repair interferes with the activation of B lymphocytes

Cells rapidly repair plasma membrane (PM) damage by a process requiring Ca²⁺-dependent lysosome exocytosis. Acid sphingomyelinase (ASM) released from lysosomes induces endocytosis of injured membrane through caveolae, membrane invaginations from lipid rafts. How B lymphocytes, lacking any known form of caveolin, repair membrane injury is unknown. Here we show that B lymphocytes repair PM wounds in a Ca²⁺-dependent manner. Wounding induces lysosome exocytosis and endocytosis of dextran and the raft-binding cholera toxin subunit B (CTB). Resealing is reduced by ASM inhibitors and ASM deficiency and enhanced or restored by extracellular exposure to sphingomyelinase. B cell activation via B cell receptors (BCRs), a process requiring lipid rafts, interferes with PM repair. Conversely, wounding inhibits BCR signaling and internalization by disrupting BCR–lipid raft coclustering and by inducing the endocytosis of raft-bound CTB separately from BCR into tubular invaginations. Thus, PM repair and B cell activation interfere with one another because of competition for lipid rafts, revealing how frequent membrane injury and repair can impair B lymphocyte–mediated immune responses.     

Lysosomes Behave as Ca2+-regulated Exocytic Vesicles in Fibroblasts and Epithelial Cells

Lysosomes are considered to be a terminal degradative compartment of the endocytic pathway, into which transport is mostly unidirectional. However, specialized secretory vesicles regulated by Ca²⁺, such as neutrophil azurophil granules, mast cell–specific granules, and cytotoxic lymphocyte lytic granules, share characteristics with lysosomes that may reflect a common biogenesis. In addition, the involvement of Ca²⁺ transients in the invasion mechanism of the parasite Trypanosoma cruzi, which occurs by fusion of lysosomes with the plasma membrane, suggested that lysosome exocytosis might be a generalized process present in most cell types.

Here we demonstrate that elevation in the intracellular free Ca²⁺ concentration of normal rat kidney (NRK) fibroblasts induces fusion of lysosomes with the plasma membrane. This was verified by measuring the release of the lysosomal enzyme β-hexosaminidase, the appearance on the plasma membrane of the lysosomal glycoprotein lgp120, the release of fluid-phase tracers previously loaded into lysosomes, and the release of the lysosomally processed form of cathepsin D. Exposure to the Ca²⁺ ionophore ionomycin or addition of Ca²⁺containing buffers to streptolysin O–permeabilized cells induced exocytosis of ~10% of the total lysosomes of NRK cells. The process was also detected in other cell types such as epithelial cells and myoblasts. Lysosomal exocytosis was found to require micromolar levels of Ca²⁺ and to be temperature and ATP dependent, similar to Ca²⁺-regulated secretory mechanisms in specialized cells.

These findings highlight a novel role for lysosomes in cellular membrane traffic and suggest that fusion of lysosomes with the plasma membrane may be an ubiquitous form of Ca²⁺-regulated exocytosis.

     

Localized topological changes of the plasma membrane upon exocytosis visualized by polarized TIRFM

Total internal reflection fluorescence microscopy (TIRFM) images the plasma membrane–cytosol interface and has allowed insights into the behavior of individual secretory granules before and during exocytosis. Much less is known about the dynamics of the other partner in exocytosis, the plasma membrane. In this study, we report the implementation of a TIRFM-based polarization technique to detect rapid submicrometer changes in plasma membrane topology as a result of exocytosis. A theoretical analysis of the technique is presented together with image simulations of predicted topologies of the postfusion granule membrane–plasma membrane complex. Experiments on diI-stained bovine adrenal chromaffin cells using polarized TIRFM demonstrate rapid and varied submicrometer changes in plasma membrane topology at sites of exocytosis that occur immediately upon fusion. We provide direct evidence for a persistent curvature in the exocytotic region that is altered by inhibition of dynamin guanosine triphosphatase activity and is temporally distinct from endocytosis measured by VMAT2-pHluorin.     

Insulin-regulated Glut4 Translocation: MEMBRANE PROTEIN TRAFFICKING WITH SIX DISTINCTIVE STEPS*

The trafficking kinetics of Glut4, the transferrin (Tf) receptor, and LRP1 were quantified in adipocytes and undifferentiated fibroblasts. Six steps were identified that determine steady state cell surface Glut4: (i) endocytosis, (ii) degradation, (iii) sorting, (iv) sequestration, (v) release, and (vi) tethering/docking/fusion. Endocytosis of Glut4 is 3 times slower than the Tf receptor in fibroblasts (k_en = 0.2 min⁻¹ versus 0.6 min⁻¹). Differentiation decreases Glut4 k_en 40% (k_en = 0.12 min⁻¹). Differentiation also decreases Glut4 degradation, increasing total and cell surface Glut4 3-fold. In fibroblasts, Glut4 is recycled from endosomes through a slow constitutive pathway (k_ex = 0.025–0.038 min⁻¹), not through the fast Tf receptor pathway (k_ex = 0.2 min⁻¹). The k_ex measured in adipocytes after insulin stimulation is similar (k_ex = 0.027 min⁻¹). Differentiation decreases the rate constant for sorting into the Glut4 recycling pathway (k_sort) 3-fold. In adipocytes, Glut4 is also sorted from endosomes into a second exocytic pathway through Glut4 storage vesicles (GSVs). Surprisingly, transfer from endosomes into GSVs is highly regulated; insulin increases the rate constant for sequestration (k_seq) 8-fold. Release from sequestration in GSVs is rate-limiting for Glut4 exocytosis in basal adipocytes. AS160 regulates this step. Tethering/docking/fusion of GSVs to the plasma membrane is regulated through an AS160-independent process. Insulin increases the rate of release and fusion of GSVs (k_fuseG) 40-fold. LRP1 cycles with the Tf receptor and Glut4 in fibroblasts but predominantly with Glut4 after differentiation. Surprisingly, AS160 knockdown accelerated LRP1 exocytosis in basal and insulin-stimulated adipocytes. These data indicate that AS160 may regulate trafficking into as well as release from GSVs.     

Role of turgor pressure in endocytosis in fission yeast

Yeast and other walled cells possess high internal turgor pressure that allows them to grow and survive in the environment. This turgor pressure, however, may oppose the invagination of the plasma membrane needed for endocytosis. Here we study the effects of turgor pressure on endocytosis in the fission yeast Schizosaccharomyces pombe by time-lapse imaging of individual endocytic sites. Decreasing effective turgor pressure by addition of sorbitol to the media significantly accelerates early steps in the endocytic process before actin assembly and membrane ingression but does not affect the velocity or depth of ingression of the endocytic pit in wild-type cells. Sorbitol also rescues endocytic ingression defects of certain endocytic mutants and of cells treated with a low dose of the actin inhibitor latrunculin A. Endocytosis proceeds after removal of the cell wall, suggesting that the cell wall does not contribute mechanically to this process. These studies suggest that endocytosis is governed by a mechanical balance between local actin-dependent inward forces and opposing forces from high internal turgor pressure on the plasma membrane.     

Exocytosis of acid sphingomyelinase by wounded cells promotes endocytosis and plasma membrane repair

Rapid plasma membrane resealing is essential for cellular survival. Earlier studies showed that plasma membrane repair requires Ca²⁺-dependent exocytosis of lysosomes and a rapid form of endocytosis that removes membrane lesions. However, the functional relationship between lysosomal exocytosis and the rapid endocytosis that follows membrane injury is unknown. In this study, we show that the lysosomal enzyme acid sphingomyelinase (ASM) is released extracellularly when cells are wounded in the presence of Ca²⁺. ASM-deficient cells, including human cells from Niemann-Pick type A (NPA) patients, undergo lysosomal exocytosis after wounding but are defective in injury-dependent endocytosis and plasma membrane repair. Exogenously added recombinant human ASM restores endocytosis and resealing in ASM-depleted cells, suggesting that conversion of plasma membrane sphingomyelin to ceramide by this lysosomal enzyme promotes lesion internalization. These findings reveal a molecular mechanism for restoration of plasma membrane integrity through exocytosis of lysosomes and identify defective plasma membrane repair as a possible component of the severe pathology observed in NPA patients.     

Physiological Control of Chloride Transport in Chara corallina: I. EFFECTS OF LOW TEMPERATURE, CELL TURGOR PRESSURE, AND ANIONS

The rate of Cl⁻ transport at the plasma membrane of the freshwater alga Chara corallina is investigated with respect to possible in vivo controls acting in addition to the two well established ones of cytoplasmic Cl⁻ and cytoplasmic pH. In contrast with results from many other plant tissues, halides appear to be the only anions capable of inhibiting Cl⁻ transport, either from the outside or inside surfaces of the plasma membrane. Cell turgor pressure was also investigated. It was found that neither the influx of Cl⁻ nor that of K⁺ or HCO₂⁻ is sensitive to turgor. Internal osmotic pressure is also insensitive to turgor, a situation contrasting with that in closely related brackish water charophytes.     

Calcineurin regulates the yeast synaptojanin Inp53/Sjl3 during membrane stress

During hyperosmotic shock, Saccharomyces cerevisiae adjusts to physiological challenges, including large plasma membrane invaginations generated by rapid cell shrinkage. Calcineurin, the Ca²⁺/calmodulin–dependent phosphatase, is normally cytosolic but concentrates in puncta and at sites of polarized growth during intense osmotic stress; inhibition of calcineurin-activated gene expression suggests that restricting its access to substrates tunes calcineurin signaling specificity. Hyperosmotic shock promotes calcineurin binding to and dephosphorylation of the PI(4,5)P₂ phosphatase synaptojanin/Inp53/Sjl3 and causes dramatic calcineurin-dependent reorganization of PI(4,5)P₂-enriched membrane domains. Inp53 normally promotes sorting at the trans-Golgi network but localizes to cortical actin patches in osmotically stressed cells. By activating Inp53, calcineurin repolarizes the actin cytoskeleton and maintains normal plasma membrane morphology in synaptojanin-limited cells. In response to hyperosmotic shock and calcineurin-dependent regulation, Inp53 shifts from associating predominantly with clathrin to interacting with endocytic proteins Sla1, Bzz1, and Bsp1, suggesting that Inp53 mediates stress-specific endocytic events. This response has physiological and molecular similarities to calcineurin-regulated activity-dependent bulk endocytosis in neurons, which retrieves a bolus of plasma membrane deposited by synaptic vesicle fusion. We propose that activation of Ca²⁺/calcineurin and PI(4,5)P₂ signaling to regulate endocytosis is a fundamental and conserved response to excess membrane in eukaryotic cells.     

Regulation of contractile vacuole formation and activity in Dictyostelium

The contractile vacuole (CV) system is the osmoregulatory organelle required for survival for many free-living cells under hypotonic conditions. We identified a new CV regulator, Disgorgin, a TBC-domain-containing protein, which translocates to the CV membrane at the late stage of CV charging and regulates CV–plasma membrane fusion and discharging. disgorgin⁻ cells produce large CVs due to impaired CV–plasma membrane fusion. Disgorgin is a specific GAP for Rab8A-GTP, which also localizes to the CV and whose hydrolysis is required for discharging. We demonstrate that Drainin, a previously identified TBC-domain-containing protein, lies upstream from Disgorgin in this pathway. Unlike Disgorgin, Drainin lacks GAP activity but functions as a Rab11A effector. The BEACH family proteins LvsA and LvsD were identified in a suppressor/enhancer screen of the disgorgin⁻ large CV phenotype and demonstrated to have distinct functions in regulating CV formation. Our studies help define the pathways controlling CV function.     

Mechanisms of Granule Membrane Recapture following Exocytosis in Intact Mast Cells

In secretory cells, several exocytosis-coupled forms of endocytosis have been proposed including clathrin-mediated endocytosis, kiss-and-run endocytosis, cavicapture, and bulk endocytosis. These forms of endocytosis can be induced under different conditions, but their detailed molecular mechanisms and functions are largely unknown. We studied exocytosis and endocytosis in mast cells with both perforated-patch and whole-cell configurations of the patch clamp technique using cell capacitance measurements in combination with amperometric serotonin detection. We found that intact mast cells exhibit an early endocytosis that follows exocytosis induced by compound 48/80. Direct observation of individual exocytic and endocytic events showed a higher percentage of capacitance flickers (27.3%) and off-steps (11.4%) in intact mast cells than in dialyzed cells (5.4% and 2.9%, respectively). Moreover, we observed a type of endocytosis of large pieces of membrane that were likely formed by cumulative fusion of several secretory granules with the cell membrane. We also identified “large-capacitance flickers” that occur after large endocytosis events. Pore conductance analysis indicated that these transient events may represent “compound cavicapture,” most likely due to the flickering of a dilated fusion pore. Using fluorescence imaging of individual exocytic and endocytic events we observed that granules can fuse to granules already fused with the plasma membrane, and then the membranes and dense cores of fused granules are internalized. Altogether, our results suggest that stimulated exocytosis in intact mast cells is followed by several forms of compensatory endocytosis, including kiss-and-run endocytosis and a mechanism for efficient retrieval of the compound membrane of several secretory granules through a single membrane fission event.     

Influence of Turgor Pressure Manipulation on Plasmalemma Transport of HCO(3) and OH in Chara corallina

A modified version of the osmotic shock technique was used to investigate HCO₃⁻ and OH⁻ transport in the alga Chara corallina. Cell turgor was brought close to zero and then restored. When turgor was reduced to near the plasmolytic point using an osmoticum, little effect was observed on H¹⁴CO₃⁻ assimilation and OH⁻ transport. However, when turgor was recovered in these cells, there was a large reduction in HCO₃⁻ and OH⁻ transport activity. In contrast, when cells were air-dried to zero turgor, and rewetted to restore turgor, no significant effect on OH⁻ transport was observed.     

Cytoplasmic calcium stimulates exocytosis in a plant secretory cell

Although exocytosis is likely to occur in plant cells, the control of this process is the subject of speculation, as no direct measurements of vesicle fusion to the plasma membrane have been made. We used the patch clamp technique to monitor the secretory activity of single aleurone protoplasts by measuring membrane capacitance (C_m), while dialyzing the cytosol with different Ca²⁺ containing solutions. Secretory activity increased with [Ca²⁺]_i ~ 1 μM. This demonstrates directly the existence of exocytosis in plant cells, and suggests that both plant and animal cells share common mechanisms (cytosolic Ca²⁺) for the control of exocytotic secretion.     

PakD,a Putative p21-Activated Protein Kinase in Dictyostelium discoideum,Regulates Actin

Proper regulation of the actin cytoskeleton is essential for cell function and ultimately for survival. Tight control of actin dynamics is required for many cellular processes, including differentiation, proliferation, adhesion, chemotaxis, endocytosis, exocytosis, and multicellular development. Here we describe a putative p21-activated protein kinase, PakD, that regulates the actin cytoskeleton in Dictyostelium discoideum. We found that cells lacking pakD are unable to aggregate and thus unable to develop. Compared to the wild type, cells lacking PakD have decreased membrane extensions, suggesting defective regulation of the actin cytoskeleton. pakD⁻ cells show poor chemotaxis toward cyclic AMP (cAMP) but normal chemotaxis toward folate, suggesting that PakD mediates some but not all chemotaxis responses. pakD⁻ cells have decreased polarity when placed in a cAMP gradient, indicating that the chemotactic defects of the pakD⁻ cells may be due to an impaired cytoskeletal response to cAMP. In addition, while wild-type cells polymerize actin in response to global stimulation by cAMP, pakD⁻ cells exhibit F-actin depolymerization under the same conditions. Taken together, the results suggest that PakD is part of a pathway coordinating F-actin organization during development.     

Resident CAPS on dense-core vesicles docks and primes vesicles for fusion

The Ca²⁺-dependent exocytosis of dense-core vesicles in neuroendocrine cells requires a priming step during which SNARE protein complexes assemble. CAPS (aka CADPS) is one of several factors required for vesicle priming; however, the localization and dynamics of CAPS at sites of exocytosis in live neuroendocrine cells has not been determined. We imaged CAPS before, during, and after single-vesicle fusion events in PC12 cells by TIRF micro­scopy. In addition to being a resident on cytoplasmic dense-core vesicles, CAPS was present in clusters of approximately nine molecules near the plasma membrane that corresponded to docked/tethered vesicles. CAPS accompanied vesicles to the plasma membrane and was present at all vesicle exocytic events. The knockdown of CAPS by shRNA eliminated the VAMP-2–dependent docking and evoked exocytosis of fusion-competent vesicles. A CAPS(ΔC135) protein that does not localize to vesicles failed to rescue vesicle docking and evoked exocytosis in CAPS-depleted cells, showing that CAPS residence on vesicles is essential. Our results indicate that dense-core vesicles carry CAPS to sites of exocytosis, where CAPS promotes vesicle docking and fusion competence, probably by initiating SNARE complex assembly.     

The tip growth apparatus of Aspergillus nidulans

Hyphal tip growth in fungi is important because of the economic and medical importance of fungi, and because it may be a useful model for polarized growth in other organisms. We have investigated the central questions of the roles of cytoskeletal elements and of the precise sites of exocytosis and endocytosis at the growing hyphal tip by using the model fungus Aspergillus nidulans. Time-lapse imaging of fluorescent fusion proteins reveals a remarkably dynamic, but highly structured, tip growth apparatus. Live imaging of SYNA, a synaptobrevin homologue, and SECC, an exocyst component, reveals that vesicles accumulate in the Spitzenkörper (apical body) and fuse with the plasma membrane at the extreme apex of the hypha. SYNA is recycled from the plasma membrane by endocytosis at a collar of endocytic patches, 1–2 μm behind the apex of the hypha, that moves forward as the tip grows. Exocytosis and endocytosis are thus spatially coupled. Inhibitor studies, in combination with observations of fluorescent fusion proteins, reveal that actin functions in exocytosis and endocytosis at the tip and in holding the tip growth apparatus together. Microtubules are important for delivering vesicles to the tip area and for holding the tip growth apparatus in position.     

Assay of dense-core vesicle exocytosis using permeabilized PC12 cells

Exocytosis plays an essential role in fundamental cellular events by secreting neurotransmitters, hormones, and cytokines. Although the minimal molecular components termed SNARE that govern membrane fusion have been identified, the precise mechanisms behind the finely-tuned regulation of exocytosis executed by many molecules in addition to the actions of SNARE remain to be fully identified. Here, we evaluated a model system for assaying catecholamine secretion from permeabilized rat pheochromocytoma PC12 cells, in which the structural integrity required was preserved adequately. Among several chemical reagents used for the cell permeabilization and freezing-thawing procedures, the treatment of cells with digitonin at concentrations of 7.5–15 μM was most suitable for the secretion assay, as it was considered to cause mild disruption of the plasma membrane, enabling free access to small molecules such as Ca²⁺ and ATP to the minimal membrane fusion machinery. No additional cytosolic proteins were required to reconstitute the secretion. In this assay model, ATP was necessary to maintain the priming state before Ca²⁺-triggered exocytosis but was not required for the Ca²⁺-triggered membrane fusion process itself. The present study provides a useful cell model for exploring novel molecules that may be implicated in exocytosis such as those playing regulatory roles in addition to the “minimal membrane fusion machinery for exocytosis”, which does not require any additional special apparatus.     

The Ca2+ Sensor Protein Swiprosin-1/EFhd2 Is Present in Neurites and Involved in Kinesin-Mediated Transport in Neurons

Swiprosin-1/EFhd2 (EFhd2) is a cytoskeletal Ca²⁺ sensor protein strongly expressed in the brain. It has been shown to interact with mutant tau, which can promote neurodegeneration, but nothing is known about the physiological function of EFhd2 in the nervous system. To elucidate this question, we analyzed EFhd2^−/−/lacZ reporter mice and showed that lacZ was strongly expressed in the cortex, the dentate gyrus, the CA1 and CA2 regions of the hippocampus, the thalamus, and the olfactory bulb. Immunohistochemistry and western blotting confirmed this pattern and revealed expression of EFhd2 during neuronal maturation. In cortical neurons, EFhd2 was detected in neurites marked by MAP2 and co-localized with pre- and post-synaptic markers. Approximately one third of EFhd2 associated with a biochemically isolated synaptosome preparation. There, EFhd2 was mostly confined to the cytosolic and plasma membrane fractions. Both synaptic endocytosis and exocytosis in primary hippocampal EFhd2^−/− neurons were unaltered but transport of synaptophysin-GFP containing vesicles was enhanced in EFhd2^−/− primary hippocampal neurons, and notably, EFhd2 inhibited kinesin mediated microtubule gliding. Therefore, we found that EFhd2 is a neuronal protein that interferes with kinesin-mediated transport.     

Dynamin and endocytosis are required for the fusion of osteoclasts and myoblasts

Cell–cell fusion is an evolutionarily conserved process that leads to the formation of multinucleated myofibers, syncytiotrophoblasts and osteoclasts, allowing their respective functions. Although cell–cell fusion requires the presence of fusogenic membrane proteins and actin-dependent cytoskeletal reorganization, the precise machinery allowing cells to fuse is still poorly understood. Using an inducible knockout mouse model to generate dynamin 1– and 2–deficient primary osteoclast precursors and myoblasts, we found that fusion of both cell types requires dynamin. Osteoclast and myoblast cell–cell fusion involves the formation of actin-rich protrusions closely associated with clathrin-mediated endocytosis in the apposed cell. Furthermore, impairing endocytosis independently of dynamin also prevented cell–cell fusion. Since dynamin is involved in both the formation of actin-rich structures and in endocytosis, our results indicate that dynamin function is central to the osteoclast precursors and myoblasts fusion process, and point to an important role of endocytosis in cell–cell fusion.