RESUMEN
Eukaryotic plasma membranes are highly compartmentalized structures. So far, only a few individual proteins that function in a wide range of cellular processes have been shown to segregate into microdomains. However, the biological roles of most microdomain-associated proteins are unknown. Here, we investigated the heterogeneity of distinct microdomains and the complexity of their coexistence. This diversity was determined in living cells of intact multicellular tissues using 20 different marker proteins from Arabidopsis thaliana, mostly belonging to the Remorin protein family. These proteins associate with microdomains at the cytosolic leaflet of the plasma membrane. We characterized these membrane domains and determined their lateral dynamics by extensive quantitative image analysis. Systematic colocalization experiments with an extended subset of marker proteins tested in 45 different combinations revealed the coexistence of highly distinct membrane domains on individual cell surfaces. These data provide valuable tools to study the lateral segregation of membrane proteins and their biological functions in living plant cells. They also demonstrate that widely used biochemical approaches such as detergent-resistant membranes cannot resolve this biological complexity of membrane compartmentalization in vivo.
RESUMEN
Remorins are well-established marker proteins for plasma membrane microdomains. They specifically localize to the inner membrane leaflet despite an overall hydrophilic amino acid composition. Here, we determined amino acids and post-translational lipidations that are required for membrane association of remorin proteins. We used a combination of cell biological and biochemical approaches to localize remorin proteins and truncated variants of those in living cells and determined S-acylation on defined residues in these proteins. S-acylation of cysteine residues in a C-terminal hydrophobic core contributes to membrane association of most remorin proteins. While S-acylation patterns differ between members of this multi-gene family, initial membrane association is mediated by protein-protein or protein-lipid interactions. However, S-acylation is not a key determinant for the localization of remorins in membrane microdomains. Although remorins bind via a conserved mechanism to the plasma membrane, other membrane-resident proteins may be involved in the recruitment of remorins into membrane domains. S-acylation probably occurs after an initial targeting of the proteins to the plasma membrane and locks remorins in this compartment. As S-acylation is a reversible post-translational modification, stimulus-dependent intracellular trafficking of these proteins can be envisioned.
Asunto(s)
Proteínas Portadoras/metabolismo , Microdominios de Membrana/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Plantas/metabolismo , Acilación , Secuencia de Aminoácidos , Sitios de Unión , Proteínas Portadoras/química , Cisteína/metabolismo , Datos de Secuencia Molecular , Mutación , Péptidos/química , Péptidos/metabolismo , Fosfoproteínas/química , Proteínas de Plantas/química , Transporte de Proteínas , Esteroles/metabolismo , Relación Estructura-Actividad , Fracciones Subcelulares/metabolismoRESUMEN
Invasion of host cells by pathogenic or mutualistic microbes requires complex molecular dialogues that often determine host survival. Although several components of the underlying signaling cascades have recently been identified and characterized, our understanding of proteins that facilitate signal transduction or assemble signaling complexes is rather sparse. Our knowledge of plant-specific remorin proteins, annotated as proteins with unknown function, has recently advanced with respect to their involvement in host-microbe interactions. Current data demonstrating that a remorin protein restricts viral movement in tomato leaves and the importance of a symbiosis-specific remorin for bacterial infection of root nodules suggest that these proteins may serve such regulatory functions. Direct interactions of other remorins with a resistance protein in Arabidopsis thaliana, and differential phosphorylation upon perception of microbial-associated molecular patterns and during expression of bacterial effector proteins, strongly underline their roles in plant defense. Furthermore, the specific subcellular localization of remorins in plasma membrane microdomains now provides the opportunity to visualize membrane rafts in living plants cells. There, remorins may oligomerize and act as scaffold proteins during early signaling events. This review summarizes current knowledge of this protein family and the potential roles of remorins in membrane rafts.
Asunto(s)
Proteínas Portadoras/metabolismo , Microdominios de Membrana/metabolismo , Interacciones Microbianas/fisiología , Fosfoproteínas/metabolismo , Proteínas de Plantas/metabolismo , Esfingolípidos/metabolismo , Esteroles/metabolismo , Nicotiana/metabolismoRESUMEN
Filopodia are finger-like actin-rich protrusions that extend from the cell surface and are important for cell-cell communication and pathogen internalization. The small size and transient nature of filopodia combined with shared usage of actin regulators within cells confounds attempts to identify filopodial proteins. Here, we used phage display phenotypic screening to isolate antibodies that alter the actin morphology of filopodia-like structures (FLS) in vitro. We found that all of the antibodies that cause shorter FLS interact with SNX9, an actin regulator that binds phosphoinositides during endocytosis and at invadopodia. In cells, we discover SNX9 at specialized filopodia in Xenopus development and that SNX9 is an endogenous component of filopodia that are hijacked by Chlamydia entry. We show the use of antibody technology to identify proteins used in filopodia-like structures, and a role for SNX9 in filopodia.
Asunto(s)
Seudópodos/metabolismo , Nexinas de Clasificación/metabolismo , Proteínas de Xenopus/metabolismo , Animales , Femenino , Células HeLa , Humanos , Masculino , Nexinas de Clasificación/genética , Proteínas de Xenopus/genética , Xenopus laevisRESUMEN
Cell surface receptors govern a multitude of signalling pathways in multicellular organisms. In plants, prominent examples are the receptor kinases FLS2 and BRI1, which activate immunity and steroid-mediated growth, respectively. Intriguingly, despite inducing distinct signalling outputs, both receptors employ common downstream signalling components, which exist in plasma membrane (PM)-localised protein complexes. An important question is thus how these receptor complexes maintain signalling specificity. Live-cell imaging revealed that FLS2 and BRI1 form PM nanoclusters. Using single-particle tracking we could discriminate both cluster populations and we observed spatiotemporal separation between immune and growth signalling platforms. This finding was confirmed by visualising FLS2 and BRI1 within distinct PM nanodomains marked by specific remorin proteins and differential co-localisation with the cytoskeleton. Our results thus suggest that signalling specificity between these pathways may be explained by the spatial separation of FLS2 and BRI1 with their associated signalling components within dedicated PM nanodomains.
Asunto(s)
Proteínas de Arabidopsis/análisis , Arabidopsis/química , Membrana Celular/química , Proteínas Quinasas/análisis , Receptores de Superficie Celular/análisis , Microscopía Intravital , Análisis Espacio-TemporalRESUMEN
Curving biological membranes establishes the complex architecture of the cell and mediates membrane traffic to control flux through subcellular compartments. Common molecular mechanisms for bending membranes are evident in different cell biological contexts across eukaryotic phyla. These mechanisms can be intrinsic to the membrane bilayer (either the lipid or protein components) or can be brought about by extrinsic factors, including the cytoskeleton. Here, we review examples of membrane curvature generation in animals, fungi, and plants. We showcase the molecular mechanisms involved and how they collaborate and go on to highlight contexts of curvature that are exciting areas of future research. Lessons from how membranes are bent in yeast and mammals give hints as to the molecular mechanisms we expect to see used by plants and protists.