Plasma Membrane Proteins In Cells Research Articles

Lipid-Based Nanoparticles Fused with Natural Killer Cell Plasma Membrane Proteins for Triple-Negative Breast Cancer Therapy.

Immunotherapy combined with chemicals and genetic engineering tools is emerging as a promising strategy to treat triple-negative breast cancer (TNBC), which is more aggressive with poorer progress than other breast cancer subtypes. In this study, lipid-based nanoparticles (LNPs) possessed an NK cell-like function that could deliver tumor-specific therapeutics and inhibit tumor growth. LNPs fused with an NK cell membrane protein system (NK-LNP) have three main features: (i) hydrophilic plasmid DNA can inhibit TNBC metastasis when encapsulated within LNPs and delivered to cells; (ii) the lipid composition of LNPs, including C18 ceramide, exhibits anticancer effects; (iii) NK cell membrane proteins are immobilized on the LNP surface, enabling targeted delivery to TNBC cells. These particles facilitate the targeted delivery of HIC1 plasmid DNA and the modulation of immune cell functions. Delivered therapeutic genes can inhibit metastasis of TNBC and then induce apoptotic cell death while targeting macrophages to promote cytokine release. The anticancer effect is expected to be applied in treating various difficult-to-treat cancers with LNP fused with NK cell plasma membrane proteins, which can simultaneously deliver therapeutic chemicals and genes.

The EXO70 inhibitor Endosidin2 alters plasma membrane protein composition in Arabidopsis roots.

To sustain normal growth and allow rapid responses to environmental cues, plants alter the plasma membrane protein composition under different conditions presumably by regulation of delivery, stability, and internalization. Exocytosis is a conserved cellular process that delivers proteins and lipids to the plasma membrane or extracellular space in eukaryotes. The octameric exocyst complex contributes to exocytosis by tethering secretory vesicles to the correct site for membrane fusion; however, whether the exocyst complex acts universally for all secretory vesicle cargo or just for specialized subsets used during polarized growth and trafficking is currently unknown. In addition to its role in exocytosis, the exocyst complex is also known to participate in membrane recycling and autophagy. Using a previously identified small molecule inhibitor of the plant exocyst complex subunit EXO70A1, Endosidin2 (ES2), combined with a plasma membrane enrichment method and quantitative proteomic analysis, we examined the composition of plasma membrane proteins in the root of Arabidopsis seedlings, after inhibition of the ES2-targetted exocyst complex, and verified our findings by live imaging of GFP-tagged plasma membrane proteins in root epidermal cells. The abundance of 145 plasma membrane proteins was significantly reduced following short-term ES2 treatments and these likely represent candidate cargo proteins of exocyst-mediated trafficking. Gene Ontology analysis showed that these proteins play diverse functions in cell growth, cell wall biosynthesis, hormone signaling, stress response, membrane transport, and nutrient uptake. Additionally, we quantified the effect of ES2 on the spatial distribution of EXO70A1 with live-cell imaging. Our results indicate that the plant exocyst complex mediates constitutive dynamic transport of subsets of plasma membrane proteins during normal root growth.

Polarized cell plasma membrane composition and protein sorting.

Epithelial cell polarization is an essential biological process, serving many physiological roles including tissue morphogenesis and wound healing. A defining feature of polarization is the separation of cell plasma membrane lipids and proteins into apical and basolateral compartments between which molecular exchange is restricted. Decades ago, the apical membrane was found to be enriched in saturated lipids, glycolipids, and cholesterol. Mechanistic hypotheses to explain the biogenesis and unique composition of the apical membrane include self-assembling membrane domains (i.e., lipid rafts), specific protein sorting motifs, and post-translational modifications mediating protein sorting. However, neither the detailed composition nor the mechanisms of protein and lipid sorting between plasma membrane domains in epithelial cells have been resolved. Particularly, the lipid profile of the basolateral membrane remains unstudied, leaving doubts about the differentiation and lipid separation of the apical and basolateral membrane. We use advanced lipidomics and imaging techniques to characterize the changes in lipid organization, membrane composition, and membrane properties during the cellular polarization process. We observe that the apical membrane is enriched in highly saturated lipids and glycolipids relative to the basolateral membrane, with apical membrane biophysical properties reflecting a raft-enriched environment. Similar to lipids, the determinants of protein sorting between apical and basolateral domains are poorly understood, with past studies focusing on either specific proteins or trafficking machinery. Importantly, the apical membrane hosts an extensive extracellular glycocalyx consisting of glycolipids, glycoproteins, and polysaccharides. The role of these glycocalyx molecules on protein sorting has not been revealed, despite a major fraction of apical proteins being glycosylated. We systematically evaluate the structural determinants of apical versus basolateral sorting with a focus on transmembrane domain features, protein raft affinity, and glycosylation. We find that these protein features cooperatively direct protein sorting to the apical membrane.

Cholesterol and M2 Rendezvous in Budding and Scission of Influenza A Virus.

The cholesterol of the host cell plasma membrane and viral M2 protein plays a crucial role in multiple stages of infection and replication of the influenza A virus. Cholesterol is required for the formation of heterogeneous membrane microdomains (or rafts) in the budozone of the host cell that serves as assembly sites for the viral components. The raft microstructures act as scaffolds for several proteins. Cholesterol may further contribute to the mechanical forces necessary for membrane scission in the last stage of budding and help to maintain the stability of the virus envelope. The M2 protein has been shown to cause membrane scission in model systems by promoting the formation of curved lipid bilayer structures that, in turn, can lead to membrane vesicles budding off or scission intermediates. Membrane remodeling by M2 is intimately linked with cholesterol as it affects local lipid composition, fluidity, and stability of the membrane. Thus, both cholesterol and M2 protein contribute to the efficient and proper release of newly formed influenza viruses from the virus-infected cells.

CCR4-NOT Complex 2-A Cofactor in Host Cell for Porcine Epidemic Diarrhea Virus Infection.

The porcine epidemic diarrhea virus (PEDV) has catastrophic impacts on the global pig industry. However, there is no consensus on the primary receptor associated with the PEDV invasion of host cells. An increasing number of studies have reported that PEDV invading host cells may require collaboration between multiple receptors and to better understand the virus-host interaction during PEDV entry, surface plasmon resonance (SPR) assays are performed to investigate relevant host factors interacting with PEDV spike-1 protein (S1) in Vero and IPEC-J2 cell membranes. Subsequently, the rabbit anti-PEDV S1 polyclonal antibody is used as bait to recognize the complexes of IPEC-J2 membrane proteins with or without PEDV infection, followed by detection using liquid chromatography with tandem mass spectrometry (LC-MS-MS). Our results show that 13 and 10 proteins interacting between the S1 protein and plasma membrane protein of Vero or IPEC-J2 can be identified. More specifically, a total of 11 differentially expressed interacting proteins were identified in IPEC-J2 membrane proteins after PEDV infection, compared to the uninfected group. Furthermore, we found that the differentially interacting protein CCR4-NOT complex 2 (CNOT2), identified in PEDV S1 with plasma membrane proteins of Vero cells, is involved in viral infection. The results show that the knockout of CNOT2 significantly inhibits PEDV replication in vitro. These data provide novel insights into the entry mechanism of PEDV.

Composition and sorting of polarized cell plasma membrane compartments

Epithelial cell polarization is an essential biological process, serving many physiological roles including tissue morphogenesis and wound healing. A defining feature of polarization is the separation of cell plasma membrane lipids and proteins into apical and basolateral compartments between which molecular exchange is restricted. Decades ago, the apical membrane was found to be enriched in saturated lipids, glycolipids, and cholesterol. Mechanistic hypotheses to explain the biogenesis and unique composition of the apical membrane include self-assembling membrane domains (i.e., lipid rafts), specific protein sorting motifs, and post-translational modifications mediating protein sorting.

Therapeutic Nanobodies Targeting Cell Plasma Membrane Transport Proteins: A High-Risk/High-Gain Endeavor.

Cell plasma membrane proteins are considered as gatekeepers of the cell and play a major role in regulating various processes. Transport proteins constitute a subclass of cell plasma membrane proteins enabling the exchange of molecules and ions between the extracellular environment and the cytosol. A plethora of human pathologies are associated with the altered expression or dysfunction of cell plasma membrane transport proteins, making them interesting therapeutic drug targets. However, the search for therapeutics is challenging, since many drug candidates targeting cell plasma membrane proteins fail in (pre)clinical testing due to inadequate selectivity, specificity, potency or stability. These latter characteristics are met by nanobodies, which potentially renders them eligible therapeutics targeting cell plasma membrane proteins. Therefore, a therapeutic nanobody-based strategy seems a valid approach to target and modulate the activity of cell plasma membrane transport proteins. This review paper focuses on methodologies to generate cell plasma membrane transport protein-targeting nanobodies, and the advantages and pitfalls while generating these small antibody-derivatives, and discusses several therapeutic nanobodies directed towards transmembrane proteins, including channels and pores, adenosine triphosphate-powered pumps and porters.

Analysis of Membrane Proteins Transport from Endosomal Compartments to Vacuoles.

Endocytosis and endosomal trafficking to vacuoles play important roles in regulating the homeostasis of plasma membrane (PM) proteins in plant cells. FREE1 (FYVE domain protein required for endosomal sorting 1) is a plant-unique component of the ESCRT (endosomal sorting complex required for transport) machinery. In free1 mutant plants, PIN-FORMED 2 (PIN2)-GFP was found to mislocalize from the PM to the tonoplast. In this chapter, we describe a detailed protocol for studying vacuolar sorting and degradation of PIN2-GFP by using T-DNA insertional mutants, dexamethasone (DEX) inducible RNAi lines, and other tools, including Fei-Mao (FM) dye staining and dark treatment. By using these methods, we illustrate the endosomal trafficking and vacuolar degradation of PIN2-GFP in plants.

Proteomic Analysis of Plasma Membrane Proteins of Antler Stem Cells Using Label-Free LC⁻MS/MS.

Deer antlers are unusual mammalian organs that can fully regenerate after annual shedding. Stem cells resident in the pedicle periosteum (PPCs) provide the main cell source for antler regeneration. Central to various cellular processes are plasma membrane proteins, but the expression of these proteins has not been well documented in antler regeneration. In the present study, plasma membrane proteins of PPCs and facial periosteal cells (FPCs) were analyzed using label-free liquid chromatography–mass spetrometry (LC–MS/MS). A total of 1739 proteins were identified. Of these proteins, 53 were found solely in the PPCs, 100 solely in the FPCs, and 1576 co-existed in both PPCs and FPCs; and 39 were significantly up-regulated in PPCs and 49 up-regulated in FPCs. In total, 226 gene ontology (GO) terms were significantly enriched from the differentially expressed proteins (DEPs). Five clusters of biological processes from these GO terms comprised responses to external stimuli, signal transduction, membrane transport, regulation of tissue regeneration, and protein modification processes. Further studies are required to demonstrate the relevancy of these DEPs in antler stem cell biology and antler regeneration.

Cell-Shaping Micropatterns for Quantitative Super-Resolution Microscopy Imaging of Membrane Mechanosensing Proteins.

Patterning cells on microcontact-printed substrates is a powerful approach to control cell morphology and introduce specific mechanical cues on a cell's molecular organization. Although global changes in cellular architectures caused by micropatterns can easily be probed with diffraction-limited optical microscopy, studying molecular reorganizations at the nanoscale demands micropatterned substrates that accommodate the optical requirements of single molecule microscopy techniques. Here, we developed a simple micropatterning strategy that provides control of cellular architectures and is optimized for nanometer accuracy single molecule tracking and three-dimensional super-resolution imaging of plasma and nuclear membrane proteins in cells. This approach, based on fibronectin microcontact printing on hydrophobic organosilane monolayers, allows evanescent wave and light-sheet microscopy of cells whilst fulfilling the stringent optical demands of point reconstruction optical microscopy. By imposing steady-state mechanical cues on cells grown in these micropatterns, we reveal nanoscale remodeling in the dynamics and the structural organizations of the nuclear envelope mechanotransducing protein emerin and of the plasma membrane mechanosensing protein caveolin-1 using single particle tracking photoactivated localization microscopy and direct stochastic optical reconstruction microscopy imaging. In addition to allowing quantitative biophysical studies of mechanoresponsive membrane proteins, this approach provides an easy means to probe mechanical regulations in cellular membranes with high optical resolution and nanometer precision.

Determining Cell-surface Expression and Endocytic Rate of Proteins in Primary Astrocyte Cultures Using Biotinylation.

Cell-surface proteins mediate a wide array of functions. In many cases, their activity is regulated by endocytic processes that modulate their levels at the plasma membrane. Here, we present detailed protocols for 2 methods that facilitate the study of such processes, both of which are based on the principle of the biotinylation of cell-surface proteins. The first is designed to allow for the semi-quantitative determination of the relative levels of a particular protein at the cell-surface. In it, the lysine residues of the plasma membrane proteins of cells are first labeled with a biotin moiety. Once the cells are lysed, these proteins may then be specifically precipitated via the use of agarose-immobilized streptavidin by exploiting the natural affinity of the latter for biotin. The proteins isolated in such a manner may then be analyzed via a standard western blotting approach. The second method provides a means of determining the endocytic rate of a particular cell-surface target over a period of time. Cell-surface proteins are first modified with a biotin derivative containing a cleavable disulfide bond. The cells are then shifted back to normal culture conditions, which causes the endocytic uptake of a proportion of biotinylated proteins. Next, the disulfide bonds of non-internalized biotin groups are reduced using the membrane-impermeable reducing agent glutathione. Via this approach, endocytosed proteins may thus be isolated and quantified with a high degree of specificity.

The basolateral vesicle sorting machinery and basolateral proteins are recruited to the site of enteropathogenic E. coli microcolony growth at the apical membrane.

Foodborne Enteropathogenic Escherichia coli (EPEC) infections of the small intestine cause diarrhea especially in children and are a major cause of childhood death in developing countries. EPEC infects the apical membrane of the epithelium of the small intestine by attaching, effacing the microvilli under the bacteria and then forming microcolonies on the cell surface. We first asked the question where on epithelial cells EPEC attaches and grows. Using models of polarized epithelial monolayers, we evaluated the sites of initial EPEC attachment to the apical membrane and found that EPEC preferentially attached over the cell-cell junctions and formed microcolonies preferentially where three cells come together at tricellular tight junctions. The ability of EPEC to adhere increased when host cell polarity was compromised yielding EPEC access to basolateral proteins. EPEC pedestals contain basolateral cytoskeletal proteins. Thus, we asked if attached EPEC causes reorganization the protein composition of the host cell plasma membrane at sites of microcolony formation. We found that EPEC microcolony growth at the apical membrane resulted in a local accumulation of basolateral plasma membrane proteins surrounding the microcolony. Basolateral marker protein aquaporin-3 localized to forming EPEC microcolonies. Components of the basolateral vesicle targeting machinery were re-routed. The Exocyst (Exo70) was recruited to individual EPEC as was the basolateral vesicle SNARE VAMP-3. Moreover, several Rab variants were also recruited to the infection site, and their dominant-negative equivalents were not. To quantitatively study the recruitment of basolateral proteins, we created a pulse of the temperature sensitive basolateral VSVG, VSVG3-SP-GFP, from the trans-Golgi Network. We found that after release from the TGN, significantly more VSVG3-SP-GFP accumulated at the site of microcolony growth than on equivalent membrane regions of uninfected cells. This suggests that trafficking of vesicles destined for the basolateral membrane are redirected to the apical site of microcolony growth. Thus, in addition to disrupting host cell fence function, local host cell plasma membrane protein composition is changed by altered protein trafficking and recruitment of basolateral proteins to the apical microcolony. This may aid EPEC attachment and subsequent microcolony growth.

Structural basis of the interaction between the putative adhesion-involved and iron-regulated FrpD and FrpC proteins of Neisseria meningitidis

The iron-regulated protein FrpD from Neisseria meningitidis is an outer membrane lipoprotein that interacts with very high affinity (Kd ~ 0.2 nM) with the N-terminal domain of FrpC, a Type I-secreted protein from the Repeat in ToXin (RTX) protein family. In the presence of Ca2+, FrpC undergoes Ca2+ -dependent protein trans-splicing that includes an autocatalytic cleavage of the Asp414-Pro415 peptide bond and formation of an Asp414-Lys isopeptide bond. Here, we report the high-resolution structure of FrpD and describe the structure-function relationships underlying the interaction between FrpD and FrpC1-414. We identified FrpD residues involved in FrpC1-414 binding, which enabled localization of FrpD within the low-resolution SAXS model of the FrpD-FrpC1-414 complex. Moreover, the trans-splicing activity of FrpC resulted in covalent linkage of the FrpC1-414 fragment to plasma membrane proteins of epithelial cells in vitro, suggesting that formation of the FrpD-FrpC1-414 complex may be involved in the interaction of meningococci with the host cell surface.

Transcriptome profile of yeast reveals the essential role of PMA2 and uncharacterized gene YBR056W-A (MNC1) in adaptation to toxic manganese concentration.

Adaptation of S. cerevisiae to toxic concentrations of manganese provides a physiological model of heavy metal homeostasis. Transcriptome analysis of adapted yeast cells reveals upregulation of cell wall and plasma membrane proteins including membrane transporters. The gene expression in adapted cells differs from that of cells under short-term toxic metal stress. Among the most significantly upregulated genes are PMA2, encoding an ortholog of Pma1 H+-ATPase of the plasma membrane, and YBR056W-A, encoding a putative membrane protein Mnc1 that belongs to the CYSTM family and presumably chelates manganese at the cell surface. We demonstrate that these genes are essential for the adaptation to toxic manganese concentration and propose an extended scheme of manganese detoxification in yeast.

Isolation of GFP-tagged plasma membrane protein from Arabidopsis egg cells.

Angiosperms possess a double fertilization system for sexual reproduction. Double fertilization is regulated by interactions among proteins localized in the plasma membrane of each sex gamete. A few plasma membrane resident proteins regulating double fertilization have been identified in male gametes. In contrast, no fertilization regulators in female gamete plasma membrane have been identified, largely due to difficulties in the isolation and collection of female gametes. We had produced Arabidopsis transgenic plant pDD45::GFP-AtPIP2;1 where the egg cell plasma membrane was specifically labeled with GFP (Igawa et al. 2013). The protein extract derived from approximately 200 pistils, which contained unfertilized and mature egg cells, was subjected to immunoprecipitation using anti-GFP antibody. As a result, both GFP and AtPIP2;1 were specifically detected in immunoprecipitated proteins from pistil tissues of pDD45::GFP-AtPIP2;1 transgenic plant, but not in those of wild type pistils. It was revealed that specific proteins expressed in the egg cells were successfully isolated from pistil cell population. The method described here showed the feasibility of isolating specific egg cell plasma membrane protein without gamete isolation and collection procedures.

Novel Biosensor of Membrane Protein Proximity Based on Fluorogen Activated Proteins.

We describe a novel biosensor system for reporting proximity between cell surface proteins in live cultured cells. The biosensor takes advantage of recently developed fluorogen-activating proteins (FAPs) that display fluorescence only when bound to otherwise-nonfluorescent fluorogen molecules. To demonstrate feasibility for the approach, two recombinant rapamycin-binding proteins were expressed as single-pass plasma membrane proteins in HeLa cells; one of the proteins (scAvd- FRB) carried an extracellular avidin tag; the other (HL1-TO1-FKBP) carried an extracellular FAP. Cells were incubated with a membrane-impermeable bivalent ligand (biotin-PEG2000-DIR) consisting of biotin joined to a dimethyl-indole red (DIR) fluorogen by a polyethylene glycol linker, thus tethering the fluorogen to the scAvd-FRB fusion protein. Addition of rapamycin, which promotes FKBP-FRB dimerization and thereby brings the FAP in close proximity to the tethered fluorogen, led to a significant increase in DIR fluorescence. We call the new proximity assay TEFLA, for tethered fluorogen assay.

Mapping the O-Mannose Glycoproteome in Saccharomyces cerevisiae

O-Mannosylation is a vital protein modification conserved from fungi to humans. Yeast is a perfect model to study this post-translational modification, because in contrast to mammals O-mannosylation is the only type of O-glycosylation. In an essential step toward the full understanding of protein O-mannosylation we mapped the O-mannose glycoproteome in baker's yeast. Taking advantage of an O-glycan elongation deficient yeast strain to simplify sample complexity, we identified over 500 O-glycoproteins from all subcellular compartments for which over 2300 O-mannosylation sites were mapped by electron-transfer dissociation (ETD)-based MS/MS. In this study, we focus on the 293 O-glycoproteins (over 1900 glycosylation sites identified by ETD-MS/MS) that enter the secretory pathway and are targets of ER-localized protein O-mannosyltransferases. We find that O-mannosylation is not only a prominent modification of cell wall and plasma membrane proteins, but also of a large number of proteins from the secretory pathway with crucial functions in protein glycosylation, folding, quality control, and trafficking. The analysis of glycosylation sites revealed that O-mannosylation is favored in unstructured regions and β-strands. Furthermore, O-mannosylation is impeded in the proximity of N-glycosylation sites suggesting the interplay of these types of post-translational modifications. The detailed knowledge of the target proteins and their O-mannosylation sites opens for discovery of new roles of this essential modification in eukaryotes, and for a first glance on the evolution of different types of O-glycosylation from yeast to mammals.

Efficient isolation and proteomic analysis of cell plasma membrane proteins in gastric cancer reveal a novel differentiation and progression related cell surface marker, R-cadherin.

Cell plasma membrane proteins, playing a crucial role in cell malignant transformation and development, were the main targets of tumor detection and therapy. In this study, CyDye/biotin double-labeling proteomic approach was adopted to profile the membrane proteome of gastric cancer cell line BGC-823 and paired immortalized gastric epithelial cell GES-1. Real-time PCR, Western blotting, and immunohistochemical staining were used to validate the differential expression of a novel identified cell surface marker R-cadherin in gastric cancer cells and tissues. Clinicopathological study and survival analysis were performed to estimate its roles in tumor progression and outcome prediction. Real-time PCR and Western blotting showed that the expression level of R-cadherin in gastric cancer were significantly lower than non-cancerous epithelial cell and tissues. Clinicopathological study indicated that R-cadherin was dominantly expressed on cell surface of normal gastric epithelium, and its expression deletion in gastric cancer tissues was associated with tumor site, differentiation, lymph node metastasis, and pTNM (chi-square test, P < 0.05). Those patients with R-cadherin positive expression displayed better overall survivals than negative expression group (log-rank test, P = 0.000). Cox multivariate survival analysis revealed lacking the expression of R-cadherin was a main independent predictor for poor clinical outcome in gastric cancer (RR = 5.680, 95 % CI 2.250–14.341, P < 0.01). We have established a fundamental membrane proteome database for gastric cancer and identified R-cadherin as a tumor differentiation and progression-related cell surface marker of gastric cancer. Lacking the expression of R-cadherin indicates poor prognosis in patients with gastric cancer.

Proteomic Analysis of Isolated Plasma Membrane Fractions from the Mammary Gland in Lactating Cows.

The mammary gland of dairy cows is a formidable lipid-synthesizing machine for lactation. This unique function depends on the activities of plasma membrane (PM) proteins in mammary cells. Little information is known about the expression profiles of PM proteins and their functions during the lactating process. This study investigated the proteome map of PM fractions of mammary gland in lactating cows using 1D-Gel-LC-MS/MS and identified 872 nonredundant proteins with 141 unknown proteins, wherein 215 were PM-associated proteins. Most of the PM-associated proteins were binding, transport, and catalytic proteins such as annexin proteins, heat shock proteins, integrins, RAS oncogene family members, and S100 calcium binding proteins. The PM-associated pathways such as caveolae-mediated endocytosis, leukocyte extravasation, aldosterone signaling in epithelial cells, and remodeling of epithelial adherens junctions were also significantly over-represented. Proteomic analysis revealed the characteristics and predicted functions of PM proteins isolated from the lactating bovine mammary gland. These results further provide experimental evidence for the presence of many proteins predicted in the annotated bovine genome. The data generated here also provide a reference for the PM-related functional research in the mammary gland of lactating cows.

Imaging the Alphavirus Exit Pathway

ABSTRACT Alphaviruses are small enveloped RNA viruses with highly organized structures that exclude host cell proteins. They contain an internal nucleocapsid and an external lattice of the viral E2 and E1 transmembrane proteins. Alphaviruses bud from the plasma membrane (PM), but the process and dynamics of alphavirus assembly and budding are poorly understood. Here we generated Sindbis viruses (SINVs) with fluorescent protein labels on the E2 envelope protein and exploited them to characterize virus assembly and budding in living cells. During virus infection, E2 became enriched in localized patches on the PM and in filopodium-like extensions. These E2-labeled patches and extensions contained all of the viral structural proteins. Correlative light and electron microscopy studies established that the patches and extensions colocalized with virus budding structures, while light microscopy showed that they excluded a freely diffusing PM marker protein. Exclusion required the interaction of the E2 protein with the capsid protein, a critical step in virus budding, and was associated with the immobilization of the envelope proteins on the cell surface. Virus infection induced two distinct types of extensions: tubulin-negative extensions that were ∼2 to 4 μm in length and excluded the PM marker, and tubulin-positive extensions that were >10 μm long, contained the PM marker, and could transfer virus particles to noninfected cells. Tubulin-positive extensions were selectively reduced in cells infected with a nonbudding SINV mutant. Together, our data support a model in which alphavirus infection induces reorganization of the PM and cytoskeleton, leading to virus budding from specialized sites. IMPORTANCE Alphaviruses are important and widely distributed human pathogens for which vaccines and antiviral therapies are urgently needed. These small highly organized viruses bud from the host cell PM. Virus assembly and budding are critical but little understood steps in the alphavirus life cycle. We developed alphaviruses with fluorescent protein tags on one of the viral membrane (envelope) proteins and used a variety of microscopy techniques to follow the envelope protein and a host cell PM protein during budding. We showed that alphavirus infection induced the formation of patches and extensions on the PM where the envelope proteins accumulate. These sites excluded other PM proteins and correlated with virus budding structures. Exclusion of PM proteins required specific interactions of the viral envelope proteins with the internal capsid protein. Together, our data indicate that alphaviruses extensively reorganize the cell surface and cytoskeleton to promote their assembly and budding.