For metastasis to occur cells must communicate with to their local environment to initiate growth and invasion. Exosomes have emerged as an important mediator of cell-to-cell signalling through the ...transfer of molecules such as mRNAs, microRNAs, and proteins between cells. Exosomes have been proposed to act as regulators of cancer progression. Here, we study the effect of exosomes on cell migration, an important step in metastasis. We performed cell migration assays, endocytosis assays, and exosome proteomic profiling on exosomes released from three breast cancer cell lines that model progressive stages of metastasis. Results from these experiments suggest: (1) exosomes promote cell migration and (2) the signal is stronger from exosomes isolated from cells with higher metastatic potentials; (3) exosomes are endocytosed at the same rate regardless of the cell type; (4) exosomes released from cells show differential enrichment of proteins with unique protein signatures of both identity and abundance. We conclude that breast cancer cells of increasing metastatic potential secrete exosomes with distinct protein signatures that proportionally increase cell movement and suggest that released exosomes could play an active role in metastasis.
Curved membranes are key features of intracellular organelles, and their generation involves dynamic protein complexes. Here we describe the fundamental mechanisms such as the hydrophobic insertion, ...scaffolding and crowding mechanisms these proteins use to produce membrane curvatures and complex shapes required to form intracellular organelles and vesicular structures involved in endocytosis and secretion. For each mechanism, we discuss its cellular functions as well as the underlying physical principles and the specific membrane properties required for the mechanism to be feasible. We propose that the integration of individual mechanisms into a highly controlled, robust process of curvature generation often relies on the assembly of proteins into coats. How cells unify and organize the curvature-generating factors at the nanoscale is presented for three ubiquitous coats central for membrane trafficking in eukaryotes: clathrin-coated pits, caveolae, and COPI and COPII coats. The emerging theme is that these coats arrange and coordinate curvature-generating factors in time and space to dynamically shape membranes to accomplish membrane trafficking within cells.
Watching biological molecules provides clues to their function and regulation. Some of the most powerful methods of labeling proteins for imaging use genetically encoded fluorescent fusion tags. ...There are four standard genetic methods of covalently tagging a protein with a fluorescent probe for cellular imaging. These use (i) autofluorescent proteins, (ii) self-labeling enzymes, (iii) enzymes that catalyze the attachment of a probe to a target sequence, and (iv) biarsenical dyes that target tetracysteine motifs. Each of these techniques has advantages and disadvantages. In this review, we cover new developments in these methods and discuss practical considerations for their use in imaging proteins inside living cells.
Clathrin-mediated endocytosis (CME) is the primary mechanism eukaryotic cells use to internalize material. New imaging tools are revealing the nanoscale structural dynamics of single clathrin-coated ...sites. Recently, it has become clear that the structure and dynamics of clathrin – flat clathrin lattices and the transition to highly curved clathrin-coated vesicles – are adaptable and can follow many paths. Thus, understanding this dynamic plasticity will lead to insights into how one molecular machine can participate in multiple pathways and adapt to changing and diverse cellular environments. Here, we review recent studies that have directly addressed this structural plasticity. We discuss the structure of lattices, how clathrin lattices form, and which proteins or biophysical factors might regulate the transition between flat and curved lattices.
CME is the primary mode in eukaryotic cells of taking up extracellular or membrane-bound material. The structural changes from flat membranes to round clathrin-coated vesicles, however, are not understood.
Flat clathrin lattices are well-documented features of plasma membranes. Only recently has it become clear that flat lattices can bend into curved clathrin vesicles in live cells.
We discuss factors encouraging flat clathrin growth in mammalian cells including membrane tension, adhesion to the substrate, bulky cargo, and membrane rigidity.
We discuss factors that could encourage curvature including membrane binding domains, disordered adaptor proteins, actin recruitment, clathrin conformational changes, post-translational modifications, energy dependent clathrin exchange, and adaptor-protein redistribution.
It is unclear if flat clathrin structures are the result of frustrated endocytosis or if they are playing an active role in signaling or adhesion.
Dozens of proteins capture, polymerize and reshape the clathrin lattice during clathrin-mediated endocytosis (CME). How or if this ensemble of proteins is organized in relation to the clathrin coat ...is unknown. Here, we map key molecules involved in CME at the nanoscale using correlative super-resolution light and transmission electron microscopy. We localize 19 different endocytic proteins (amphiphysin1, AP2, β2-arrestin, CALM, clathrin, DAB2, dynamin2, EPS15, epsin1, epsin2, FCHO2, HIP1R, intersectin, NECAP, SNX9, stonin2, syndapin2, transferrin receptor, VAMP2) on thousands of individual clathrin structures, generating a comprehensive molecular architecture of endocytosis with nanoscale precision. We discover that endocytic proteins distribute into distinct spatial zones in relation to the edge of the clathrin lattice. The presence or concentrations of proteins within these zones vary at distinct stages of organelle development. We propose that endocytosis is driven by the recruitment, reorganization and loss of proteins within these partitioned nanoscale zones.
The plasma membrane separates a cell from its external environment. All materials and signals that enter or leave the cell must cross this hydrophobic barrier. Understanding the architecture and ...dynamics of the plasma membrane has been a central focus of general cellular physiology. Both light and electron microscopy have been fundamental in this endeavor and have been used to reveal the dense, complex, and dynamic nanoscale landscape of the plasma membrane. Here, I review classic and recent developments in the methods used to image and study the structure of the plasma membrane, particularly light, electron, and correlative microscopies. I will discuss their history and use for mapping the plasma membrane and focus on how these tools have provided a structural framework for understanding the membrane at the scale of molecules. Finally, I will describe how these studies provide a roadmap for determining the nanoscale architecture of other organelles and entire cells in order to bridge the gap between cellular form and function.
Protein coats, important for vesicular trafficking in eukaryotic cells, help shape membranes and package cargo. But their dynamic construction cannot be fully understood until the distinct steps of ...their assembly in their native intracellular context at molecular resolution can be visualized. For this, correlative light and electron microscopy (CLEM) is an essential tool. Here, we discuss how emerging CLEM techniques have been used to study the assembly of protein coats inside cells. We review how current and developing CLEM technologies are poised to answer fundamental questions of protein coat architecture at the nanoscale.
Spines are tiny nanoscale protrusions from dendrites of neurons. In the cortex and hippocampus, most of the excitatory postsynaptic sites reside in spines. The bulbous spine head is connected to the ...dendritic shaft by a thin membranous neck. Because the neck is narrow, spine heads are thought to function as biochemically independent signaling compartments. Thus, dynamic changes in the composition, distribution, mobility, conformations, and signaling properties of molecules contained within spines can account for much of the molecular basis of postsynaptic function and regulation. A major factor in controlling these changes is the diffusional properties of proteins within this small compartment. Advances in measurement techniques using fluorescence microscopy now make it possible to measure molecular diffusion within single dendritic spines directly. Here, we review the regulatory mechanisms of diffusion in spines by local intra-spine architecture and discuss their implications for neuronal signaling and synaptic plasticity.
Caveolae are 70-100 nm diameter plasma membrane invaginations found in abundance in adipocytes, endothelial cells, myocytes, and fibroblasts. Their bulb-shaped membrane domain is characterized and ...formed by specific lipid binding proteins including Caveolins, Cavins, Pacsin2, and EHD2. Likewise, an enrichment of cholesterol and other lipids makes caveolae a distinct membrane environment that supports proteins involved in cell-type specific signaling pathways. Their ability to detach from the plasma membrane and move through the cytosol has been shown to be important for lipid trafficking and metabolism. Here, we review recent concepts in caveolae trafficking and dynamics. Second, we discuss how ATP and GTP-regulated proteins including dynamin and EHD2 control caveolae behavior. Throughout, we summarize the potential physiological and cell biological roles of caveolae internalization and trafficking and highlight open questions in the field and future directions for study.
The GFP-based superecliptic pHluorin (SEP) enables detection of exocytosis and endocytosis, but its performance has not been duplicated in red fluorescent protein scaffolds. Here we describe ..."semisynthetic" pH-sensitive protein conjugates with organic fluorophores, carbofluorescein, and Virginia Orange that match the properties of SEP. Conjugation to genetically encoded self-labeling tags or antibodies allows visualization of both exocytosis and endocytosis, constituting new bright sensors for these key steps of synaptic transmission.
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