Multiple roles for the actin cytoskeleton during regulated exocytosis

Regulated exocytosis is the main mechanism utilized by specialized secretory cells to deliver molecules to the cell surface by virtue of membranous containers (i.e. secretory vesicles). The process involves a series of highly coordinated and sequential steps, which include the biogenesis of the vesicles, their delivery to the cell periphery, their fusion with the plasma membrane and the release of their content into the extracellular space. Each of these steps is regulated by the actin cytoskeleton. In this review, we summarize the current knowledge regarding the involvement of actin and its associated molecules during each of the exocytic steps in vertebrates, and suggest that the overall role of the actin cytoskeleton during regulated exocytosis is linked to the architecture and the physiology of the secretory cells under examination. Specifically, in neurons, neuroendocrine, endocrine, and hematopoietic cells, which contain small secretory vesicles that undergo rapid exocytosis (on the order of milliseconds), the actin cytoskeleton plays a role in pre-fusion events, where it acts primarily as a functional barrier and facilitates docking. In exocrine and other secretory cells, which contain large secretory vesicles that undergo slow exocytosis (seconds to minutes), the actin cytoskeleton plays a role in post-fusion events, where it regulates the dynamics of the fusion pore, facilitates the integration of the vesicles into the plasma membrane, provides structural support, and promotes the expulsion of large cargo molecules

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level. In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton

Endosomes Derived from Clathrin-Independent Endocytosis Serve as Precursors for Endothelial Lumen Formation

<div><p>Clathrin-independent endocytosis (CIE) is a form of bulk plasma membrane (PM) endocytosis that allows cells to sample and evaluate PM composition. Once in endosomes, the internalized proteins and lipids can be recycled back to the PM or delivered to lysosomes for degradation. Endosomes arising from CIE contain lipid and signaling molecules suggesting that they might be involved in important biological processes. During vasculogenesis, new blood vessels are formed from precursor cells in a process involving internalization and accumulation of endocytic vesicles. Here, we found that CIE has a role in endothelial lumen formation. Specifically, we found that human vascular endothelial cells (HUVECs) utilize CIE for internalization of distinct cargo molecules and that in three-dimensional cultures CIE membranes are delivered to the newly formed lumen. </p> </div

CIE and CME occur in HUVECs.

<p>Cells were incubated with an antibody against MHCI and 594-labeled transferrin for 30 min. Internalized MHCI was visualized with 488-labeled secondary antibodies. MHCI and transferrin (Tfn) internalized in distinct carriers (inset) but were observed co-localized in the perinuclear region (arrowheads). (B) Internalization of MHCI antibody (upper panel) or CD98 antibody (lower panel) for 30 min was performed in HUVECs. After fixation, cells were labeled with the EEA1 antibody as described in Materials and Methods. Internalized MHCI and CD98 both reached an EEA1 positive endosome. Bar, 10 μm.</p

Internalized MHCI and CD98 co-localize with fluid phase markers in HUVECs.

<p>HUVECs were plated in three-dimensional collagen gels and allowed to internalize rhodamine-dextran and either Alexa-647 conjugated antibody against MHCI or FITC conjugated antibody against CD98 for 1 h. Cells were digested out of the collagen and plated onto poly-L-lysine coated coverslips. Pinocytic vesicles labeled with rhodamine dextran were obvious after 1 h of internalization and many of them were also positive for MHCI or CD98. Bar, 10 μm.</p