RESUMEN
Forces generated by actin assembly assist membrane invagination during clathrin-mediated endocytosis (CME). The sequential recruitment of core endocytic proteins and regulatory proteins, and assembly of the actin network, are well documented in live cells and are highly conserved from yeasts to humans. However, understanding of CME protein self-organization, as well as the biochemical and mechanical principles that underlie actin's role in CME, is lacking. Here, we show that supported lipid bilayers coated with purified yeast Wiskott Aldrich Syndrome Protein (WASP), an endocytic actin assembly regulator, and incubated in cytoplasmic yeast extracts, recruit downstream endocytic proteins and assemble actin networks. Time-lapse imaging of WASP-coated bilayers revealed sequential recruitment of proteins from different endocytic modules, faithfully replicating in vivo behavior. Reconstituted actin networks assemble in a WASP-dependent manner and deform lipid bilayers, as seen by electron microscopy. Time-lapse imaging revealed that vesicles are released from the lipid bilayers with a burst of actin assembly. Actin networks pushing on membranes have previously been reconstituted; here, we have reconstituted a biologically important variation of these actin networks that self-organize on bilayers and produce pulling forces sufficient to bud off membrane vesicles. We propose that actin-driven vesicle generation may represent an ancient evolutionary precursor to diverse vesicle forming processes adapted for a wide array of cellular environments and applications.
Asunto(s)
Actinas , Membrana Dobles de Lípidos , Actinas/metabolismo , Clatrina/metabolismo , Endocitosis , Saccharomyces cerevisiaeRESUMEN
The budding yeast Saccharomyces cerevisiae has a closed mitosis in which the mitotic spindle and the cytoplasmic microtubules (MTs), both of which generate forces to faithfully segregate chromosomes, remain separated by the nuclear envelope throughout the cell cycle. Kar3, the yeast kinesin-14, has distinct functions on MTs in each compartment. Here, we show that two proteins, Cik1 and Vik1, which form heterodimers with Kar3, regulate its localization and function within the cell, and along MTs in a cell cycle-dependent manner. Using a yeast MT dynamics reconstitution assay in lysates from cell cycle-synchronized cells, we found that Kar3-Vik1 induces MT catastrophes in S phase and metaphase, and limits MT polymerization in G1 and anaphase. In contrast, Kar3-Cik1 promotes catastrophes and pauses in G1, while increasing catastrophes in metaphase and anaphase. Adapting this assay to track MT motor protein motility, we observed that Cik1 is necessary for Kar3 to track MT plus-ends in S phase and metaphase but, surprisingly, not during anaphase. These experiments demonstrate how the binding partners of Kar3 modulate its diverse functions both spatially and temporally.
Asunto(s)
Cinesinas , Saccharomyces cerevisiae , Cinesinas/genética , Ciclo Celular , Anafase , MetafaseRESUMEN
Fusion-associated small transmembrane (FAST) proteins are a diverse family of nonstructural viral proteins. Once expressed on the plasma membrane of infected cells, they drive fusion with neighboring cells, increasing viral spread and pathogenicity. Unlike viral fusogens with tall ectodomains that pull two membranes together through conformational changes, FAST proteins have short fusogenic ectodomains that cannot bridge the intermembrane gap between neighboring cells. One orthoreovirus FAST protein, p14, has been shown to hijack the actin cytoskeleton to drive cell-cell fusion, but the actin adaptor-binding motif identified in p14 is not found in any other FAST protein. Here, we report that an evolutionarily divergent FAST protein, p22 from aquareovirus, also hijacks the actin cytoskeleton but does so through different adaptor proteins, Intersectin-1 and Cdc42, that trigger N-WASP-mediated branched actin assembly. We show that despite using different pathways, the cytoplasmic tail of p22 can replace that of p14 to create a potent chimeric fusogen, suggesting they are modular and play similar functional roles. When we directly couple p22 with the parallel filament nucleator formin instead of the branched actin nucleation promoting factor N-WASP, its ability to drive fusion is maintained, suggesting that localized mechanical pressure on the plasma membrane coupled to a membrane-disruptive ectodomain is sufficient to drive cell-cell fusion. This work points to a common biophysical strategy used by FAST proteins to push rather than pull membranes together to drive fusion, one that may be harnessed by other short fusogens responsible for physiological cell-cell fusion.
Asunto(s)
Actinas/metabolismo , Proteínas de la Fusión de la Membrana/metabolismo , Fusión de Membrana/fisiología , Citoesqueleto de Actina/metabolismo , Secuencia de Aminoácidos/genética , Animales , Evolución Biológica , Fusión Celular/métodos , Línea Celular , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Evolución Molecular , Humanos , Orthoreovirus/genética , Unión Proteica/genética , Reoviridae/genética , Proteínas Virales de Fusión/química , Proteínas Virales de Fusión/metabolismo , Proteínas no Estructurales Virales/metabolismo , Internalización del VirusRESUMEN
Some organelles cannot be synthesized anew, so they are segregated into daughter cells during cell division. In Saccharomyces cerevisiae, daughter cells bud from mother cells and are populated by organelles inherited from the mothers. To determine whether this organelle inheritance occurs in a stereotyped manner, we tracked organelles using fluorescence microscopy. We describe a program for organelle inheritance in budding yeast. The cortical endoplasmic reticulum (ER) and peroxisomes are inherited concomitantly with bud emergence. Next, vacuoles are inherited in small buds, followed closely by mitochondria. Finally, the nucleus and perinuclear ER are inherited when buds have nearly reached their maximal size. Because organelle inheritance timing correlates with bud morphology, which is coupled to the cell cycle, we tested whether disrupting the cell cycle alters organelle inheritance order. By arresting cell cycle progression but allowing continued bud growth, we determined that organelle inheritance still occurs when DNA replication is blocked, and that the general inheritance order is maintained. Thus, organelle inheritance follows a preferred order during polarized cell division and does not require completion of S-phase.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Ciclo Celular/genética , División Celular/genética , Peroxisomas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Surface topography profoundly influences cell adhesion, differentiation, and stem cell fate control. Numerous studies using a variety of materials demonstrate that nanoscale topographies change the intracellular organization of actin cytoskeleton and therefore a broad range of cellular dynamics in live cells. However, the underlying molecular mechanism is not well understood, leaving why actin cytoskeleton responds to topographical features unexplained and therefore preventing researchers from predicting optimal topographic features for desired cell behavior. Here we demonstrate that topography-induced membrane curvature plays a crucial role in modulating intracellular actin organization. By inducing precisely controlled membrane curvatures using engineered vertical nanostructures as topographies, we find that actin fibers form at the sites of nanostructures in a curvature-dependent manner with an upper limit for the diameter of curvature at â¼400 nm. Nanotopography-induced actin fibers are branched actin nucleated by the Arp2/3 complex and are mediated by a curvature-sensing protein FBP17. Our study reveals that the formation of nanotopography-induced actin fibers drastically reduces the amount of stress fibers and mature focal adhesions to result in the reorganization of actin cytoskeleton in the entire cell. These findings establish the membrane curvature as a key linkage between surface topography and topography-induced cell signaling and behavior.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Forma de la Célula , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , NanoestructurasRESUMEN
Microtubules (MTs) are important for cellular structure, transport of cargoes and segregation of chromosomes and organelles during mitosis. The stochastic growth and shrinkage of MTs, known as dynamic instability, is necessary for these functions. Previous studies to determine how individual MT-associated proteins (MAPs) affect MT dynamics have been performed either through in vivo studies, which provide limited opportunity for observation of individual MTs or manipulation of conditions, or in vitro studies, which focus either on purified proteins, and therefore lack cellular complexity, or on cell extracts made from genetically intractable organisms. In order to investigate the ensemble activities of all MAPs on MT dynamics using lysates made from a genetically tractable organism, we developed a cell-free assay for budding yeast lysates using total internal reflection fluorescence (TIRF) microscopy. Lysates were prepared from yeast strains expressing GFP-tubulin. MT polymerization from pre-assembled MT seeds adhered to a coverslip was observed in real time. Through use of cell division cycle (cdc) and MT depolymerase mutants, we found that MT polymerization and dynamic instability are dependent on the cell cycle state and the activities of specific MAPs.
Asunto(s)
Microtúbulos/metabolismo , Mitosis/fisiología , Tubulina (Proteína)/metabolismo , Cinesinas/metabolismo , Microscopía Fluorescente/métodos , Proteínas Asociadas a Microtúbulos/metabolismo , Polimerizacion , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomycetales/metabolismoRESUMEN
A critical step in cellular-trafficking pathways is the budding of membranes by protein coats, which recent experiments have demonstrated can be inhibited by elevated membrane tension. The robustness of processes like clathrin-mediated endocytosis (CME) across a diverse range of organisms and mechanical environments suggests that the protein machinery in this process has evolved to take advantage of some set of physical design principles to ensure robust vesiculation against opposing forces like membrane tension. Using a theoretical model for membrane mechanics and membrane protein interaction, we have systematically investigated the influence of membrane rigidity, curvature induced by the protein coat, area covered by the protein coat, membrane tension, and force from actin polymerization on bud formation. Under low tension, the membrane smoothly evolves from a flat to budded morphology as the coat area or spontaneous curvature increases, whereas the membrane remains essentially flat at high tensions. At intermediate, physiologically relevant, tensions, the membrane undergoes a "snap-through instability" in which small changes in the coat area, spontaneous curvature or membrane tension cause the membrane to "snap" from an open, U-shape to a closed bud. This instability can be smoothed out by increasing the bending rigidity of the coat, allowing for successful budding at higher membrane tensions. Additionally, applied force from actin polymerization can bypass the instability by inducing a smooth transition from an open to a closed bud. Finally, a combination of increased coat rigidity and force from actin polymerization enables robust vesiculation even at high membrane tensions.
Asunto(s)
Membrana Celular/química , Vesículas Cubiertas por Clatrina/fisiología , Clatrina/fisiología , Simulación por Computador , Endocitosis/fisiología , Proteínas de la Membrana/fisiología , Modelos Químicos , Estrés Mecánico , Algoritmos , Fenómenos Biomecánicos , Membrana Celular/ultraestructura , Fluidez de la Membrana , Proteínas de la Membrana/química , Propiedades de SuperficieRESUMEN
Clathrin-mediated endocytosis is an essential cellular process that involves the concerted assembly and disassembly of many different proteins at the plasma membrane. In yeast, live-cell imaging has shown that the spatiotemporal dynamics of these proteins is highly stereotypical. Recent work has focused on determining how the timing and functions of endocytic proteins are regulated. In this Cell Science at a Glance article and accompanying poster, we review our current knowledge of the timeline of endocytic site maturation and discuss recent works focusing on how phosphorylation, ubiquitylation and lipids regulate various aspects of the process.
Asunto(s)
Membrana Celular/metabolismo , Clatrina/metabolismo , Endocitosis , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomycetales/fisiología , Animales , Humanos , Metabolismo de los Lípidos , Fosforilación , UbiquitinaciónRESUMEN
Assembly of appropriately oriented actin cables nucleated by formin proteins is necessary for many biological processes in diverse eukaryotes. However, compared with knowledge of how nucleation of dendritic actin filament arrays by the actin-related protein-2/3 complex is regulated, the in vivo regulatory mechanisms for actin cable formation are less clear. To gain insights into mechanisms for regulating actin cable assembly, we reconstituted the assembly process in vitro by introducing microspheres functionalized with the C terminus of the budding yeast formin Bni1 into extracts prepared from yeast cells at different cell-cycle stages. EM studies showed that unbranched actin filament bundles were reconstituted successfully in the yeast extracts. Only extracts enriched in the mitotic cyclin Clb2 were competent for actin cable assembly, and cyclin-dependent kinase 1 activity was indispensible. Cyclin-dependent kinase 1 activity also was found to regulate cable assembly in vivo. Here we present evidence that formin cell-cycle regulation is conserved in vertebrates. The use of the cable-reconstitution system to test roles for the key actin-binding proteins tropomyosin, capping protein, and cofilin provided important insights into assembly regulation. Furthermore, using mass spectrometry, we identified components of the actin cables formed in yeast extracts, providing the basis for comprehensive understanding of cable assembly and regulation.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Ciclo Celular/fisiología , Proteínas de Microfilamentos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Citoesqueleto de Actina/ultraestructura , Western Blotting , Proteína Quinasa CDC2/metabolismo , Procesamiento de Imagen Asistido por Computador , Imagenología Tridimensional , Espectrometría de Masas , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Microesferas , Polimerizacion , Saccharomyces cerevisiaeRESUMEN
The chromosomal passenger complex (CPC) is a key regulator of mitosis in eukaryotes. It comprises four essential and conserved proteins known in mammals/yeasts as Aurora B/Ipl1, INCENP/Sli15, Survivin/Bir1, and Borealin/Nbl1. These subunits act together in a highly controlled fashion. Regulation of Aurora B/Ipl1 kinase activity and localization is critical for CPC function. Although regulation of CPC localization and kinase activity in vivo has been investigated elsewhere, studies on the complete, four-subunit CPC and its basic biochemical properties are only beginning. Here we describe the biochemical characterization of purified and complete Saccharomyces cerevisiae four-subunit CPC. We determined the affinity of the CPC for microtubules and demonstrated that the binding of CPC to microtubules is primarily electrostatic in nature and depends on the acidic C-terminal tail (E-hook) of tubulin. Moreover, phosphorylation of INCENP/Sli15 on its microtubule binding region also negatively regulates CPC affinity for microtubules. Furthermore, we show that phosphorylation of INCENP/Sli15 is required for activation of the kinase Aurora B/Ipl1 and can occur in trans. Although phosphorylation of INCENP/Sli15 is essential for activation, we determined that a version of the CPC lacking the INCENP/Sli15 microtubule binding region (residues Glu-91 to Ile-631) is able to form an intact complex that retains microtubule binding activity. Thus, we conclude that this INCENP/Sli15 linker domain plays a largely regulatory function and is not essential for complex formation or microtubule binding.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Complejos Multiproteicos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Aurora Quinasas , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas Asociadas a Microtúbulos/genética , Microtúbulos/genética , Complejos Multiproteicos/genética , Fosforilación/fisiología , Proteínas Serina-Treonina Quinasas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Anionic phospholipids PI(4,5)P2 and phosphatidylserine (PS) are enriched in the cytosolic leaflet of the plasma membrane where endocytic sites form. In this study, we investigated the roles of PI(4,5)P2 and PS in clathrin-mediated endocytosis (CME) site initiation and vesicle formation in Saccharomyces cerevisiae. Live-cell imaging of endocytic protein dynamics in an mss4(ts) mutant, which has severely reduced PI(4,5)P2 levels, revealed that PI(4,5)P2 is required for endocytic membrane invagination but is less important for endocytic site initiation. We also demonstrated that, in various deletion mutants of genes encoding components of the Rcy1-Ypt31/32 GTPase pathway, endocytic proteins dynamically assemble not only on the plasma membrane but also on intracellular membrane compartments, which are likely derived from early endosomes. In rcy1Δ cells, fluorescent biosensors indicated that PI(4,5)P2 only localized to the plasma membrane while PS localized to both the plasma membrane and intracellular membranes. Furthermore, we found that polarized endocytic patch establishment is defective in the PS-deficient cho1Δ mutant. We propose that PS is important for directing endocytic proteins to the plasma membrane and that PI(4,5)P2 is required to facilitate endocytic membrane invagination.
Asunto(s)
Clatrina/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatidilserinas/metabolismo , Transporte Biológico , Membrana Celular/metabolismo , Endocitosis , Proteínas F-Box/metabolismo , Membranas Intracelulares/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Src homology 3 (SH3) domains bind peptides to mediate protein-protein interactions that assemble and regulate dynamic biological processes. We surveyed the repertoire of SH3 binding specificity using peptide phage display in a metazoan, the worm Caenorhabditis elegans, and discovered that it structurally mirrors that of the budding yeast Saccharomyces cerevisiae. We then mapped the worm SH3 interactome using stringent yeast two-hybrid and compared it with the equivalent map for yeast. We found that the worm SH3 interactome resembles the analogous yeast network because it is significantly enriched for proteins with roles in endocytosis. Nevertheless, orthologous SH3 domain-mediated interactions are highly rewired. Our results suggest a model of network evolution where general function of the SH3 domain network is conserved over its specific form.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Dominios Homologos src/genética , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Secuencia Conservada , Endocitosis/genética , Evolución Molecular , Datos de Secuencia Molecular , Mapeo de Interacción de Proteínas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Homología Estructural de Proteína , Técnicas del Sistema de Dos HíbridosRESUMEN
During endocytic vesicle formation, distinct subdomains along the membrane invagination are specified by different proteins, which bend the membrane and drive scission. Bin-Amphiphysin-Rvs (BAR) and Fer-CIP4 homology-BAR (F-BAR) proteins can induce membrane curvature and have been suggested to facilitate membrane invagination and scission. Two F-BAR proteins, Syp1 and Bzz1, are found at budding yeast endocytic sites. Syp1 arrives early but departs from the endocytic site before formation of deep membrane invaginations and scission. Using genetic, spatiotemporal, and ultrastructural analyses, we demonstrate that Bzz1, the heterodimeric BAR domain protein Rvs161/167, actin polymerization, and the lipid phosphatase Sjl2 cooperate, each through a distinct mechanism, to induce membrane scission in yeast. Additionally, actin assembly and Rvs161/167 cooperate to drive formation of deep invaginations. Finally, we find that Bzz1, acting at the invagination base, stabilizes endocytic sites and functions with Rvs161/167, localized along the tubule, to achieve proper endocytic membrane geometry necessary for efficient scission. Together, our results reveal that dynamic interplay between a lipid phosphatase, actin assembly, and membrane-sculpting proteins leads to proper membrane shaping, tubule stabilization, and scission.
Asunto(s)
Endocitosis , Proteínas de la Membrana/metabolismo , Secuencia de Aminoácidos , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Proteínas de la Membrana/química , Datos de Secuencia Molecular , Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
During clathrin-mediated endocytosis (CME), dozens of proteins are recruited to nascent CME sites on the plasma membrane. Coordination of endocytic protein recruitment in time and space is important for efficient CME. Here, we show that the multivalent scaffold protein intersectin1 (ITSN1) promotes CME by organizing and stabilizing endocytic protein interaction networks. By live-cell imaging of genome-edited cells, we observed that endogenously labeled ITSN1 is recruited to CME sites shortly after they begin to assemble. Knocking down ITSN1 impaired endocytic protein recruitment during the stabilization stage of CME site assembly. Artificially locating ITSN1 to the mitochondria surface was sufficient to assemble puncta consisting of CME initiation proteins, including EPS15, FCHO, adaptor proteins, the AP2 complex and epsin1 (EPN1), and the vesicle scission GTPase dynamin2 (DNM2). ITSN1 can form puncta and recruit DNM2 independently of EPS15/FCHO or EPN1. Our work redefines ITSN1's primary endocytic role as organizing and stabilizing the CME protein interaction networks rather than a previously suggested role in initiation and provides new insights into the multi-step and multi-zone organization of CME site assembly.
RESUMEN
The myosin-Is, Myo3 and Myo5 in budding yeast, are implicated in force generation and actin assembly during clathrin-mediated endocytosis (CME). The myosin-Is have motor activity, bind the plasma membrane, and activate the Arp2/3 complex to promote branched actin assembly. We reveal that Myo5 's force-generating motor activity and nucleation-promoting factor (NPF) activity each must be coupled to membrane binding for successful CME. However, the motor and NPF activities are modular and separable, showing that these activities function independently rather than in an obligatorily integrated manner to provide myosin-I's essential functions in actin network assembly and force generation during budding yeast CME.
RESUMEN
The high turgor pressure across the plasma membrane of yeasts creates a requirement for substantial force production by actin polymerization and myosin motor activity for clathrin-mediated endocytosis (CME). Endocytic internalization is severely impeded in the absence of fimbrin, an actin filament crosslinking protein called Sac6 in budding yeast. Here, we combine live-cell imaging and mathematical modeling to gain new insights into the role of actin filament crosslinking proteins in force generation. Genetic manipulation showed that CME sites with more crosslinking proteins are more effective at internalization under high load. Simulations of an experimentally constrained, agent-based mathematical model recapitulate the result that endocytic networks with more double-bound fimbrin molecules internalize the plasma membrane against elevated turgor pressure more effectively. Networks with large numbers of crosslinks also have more growing actin filament barbed ends at the plasma membrane, where the addition of new actin monomers contributes to force generation and vesicle internalization. Our results provide a richer understanding of the crucial role played by actin filament crosslinking proteins during actin network force generation, highlighting the contribution of these proteins to the self-organization of the actin filament network and force generation under increased load.
RESUMEN
The HIV-1 accessory protein Nef hijacks clathrin adaptors to degrade or mislocalize host proteins involved in antiviral defenses. Here, using quantitative live-cell microscopy in genome-edited Jurkat cells, we investigate the impact of Nef on clathrin-mediated endocytosis (CME), a major pathway for membrane protein internalization in mammalian cells. Nef is recruited to CME sites on the plasma membrane, and this recruitment is associated with an increase in the recruitment and lifetime of the CME coat protein AP-2 and the late-arriving CME protein dynamin2. Furthermore, we find that CME sites that recruit Nef are more likely to recruit dynamin2 and transferrin, suggesting that Nef recruitment to CME sites promotes site maturation to ensure high efficiency in host protein downregulation. Implications of these observations for HIV-1 infection are discussed.
Asunto(s)
Clatrina , Endocitosis , VIH-1 , Productos del Gen nef del Virus de la Inmunodeficiencia Humana , Animales , Humanos , Membrana Celular/metabolismo , Clatrina/metabolismo , Endocitosis/fisiología , VIH-1/metabolismo , Células Jurkat , Proteínas de la Membrana/metabolismo , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
The Arp2/3 complex, which is crucial for actin-based motility, nucleates actin filaments and organizes them into y-branched networks. The Arp2 subunit has been shown to hydrolyse ATP, but the functional importance of Arp2/3 ATP hydrolysis is not known. Here, we analysed an Arp2 mutant in Saccharomyces cerevisiae that is defective in ATP hydrolysis. Arp2 ATP hydrolysis and Arp2/3-dependent actin nucleation occur almost simultaneously. However, ATP hydrolysis is not required for nucleation. In addition, Arp2 ATP hydrolysis is not required for the release of a WASP-like activator from y-branches. ATP hydrolysis by Arp2, and possibly Arp3, is essential for efficient y-branch dissociation in vitro. In living cells, both Arp2 and Arp3 ATP-hydrolysis mutants exhibit defects in endocytic internalization and actin-network disassembly. Our results suggest a critical feature of dendritic nucleation in which debranching and subsequent actin-filament remodelling and/or depolymerization are important for endocytic vesicle morphogenesis.
Asunto(s)
Proteína 2 Relacionada con la Actina/metabolismo , Proteína 3 Relacionada con la Actina/metabolismo , Actinas/metabolismo , Adenosina Trifosfato/metabolismo , Endocitosis/fisiología , Proteína 2 Relacionada con la Actina/genética , Proteína 3 Relacionada con la Actina/genética , Catálisis , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Hidrólisis , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Microscopía Fluorescente , Modelos Biológicos , Mutación/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Defining the ultrastructure of endocytic sites and localization of endocytic proteins in Saccharomyces cerevisiae by immunoelectron microscopy is central in understanding the mechanisms of membrane deformation and scission during endocytosis. We show that an improved sample preparation protocol based on high-pressure freezing, freeze substitution, and low-temperature embedding allows us to maintain the cellular fine structure and to immunolabel green fluorescent protein-tagged endocytic proteins or actin in the same sections. Using this technique we analyzed the stepwise deformation of endocytic membranes and immunolocalized the endocytic proteins Abp1p, Sla1p, Rvs167p, and actin, and were able to draw a clear ultrastructural distinction between endocytic sites and eisosomes by immunolocalizing Pil1p. In addition to defining the geometry and the fine structure of budding yeast endocytic sites, we observed associated actin filaments forming a cage-like meshwork around the endocytic membrane.
Asunto(s)
Endocitosis , Microscopía Electrónica de Transmisión/métodos , Microscopía Inmunoelectrónica/métodos , Saccharomyces cerevisiae/ultraestructura , Actinas/metabolismo , Proteínas del Citoesqueleto/metabolismo , Substitución por Congelación , Saccharomyces cerevisiae/fisiología , Proteínas de Saccharomyces cerevisiae/metabolismo , Adhesión del TejidoRESUMEN
An increasing corpus of research has demonstrated that membrane shape, generated either by the external environment of the cell or by intrinsic mechanisms such as cytokinesis and vesicle or organelle formation, is an important parameter in the control of diverse cellular processes. In this review we discuss recent findings that demonstrate how membrane curvature (from nanometer to micron length-scales) alters protein function. We describe an expanding toolkit for experimentally modulating membrane curvature to reveal effects on protein function, and discuss how membrane curvature - far from being a passive consequence of the physical environment and the internal protein activity of a cell - is an important signal that controls protein affinity and enzymatic activity to ensure robust forward progression of key processes within the cell.