The Role of Actin Cytoskeleton in Endocytosis and Exocytosis in the Salivary Glands of Live Rodents

In the last two decades, mammalian cell biology has greatly benefited from major technological advances in light microscopy that have enabled imaging virtually any cellular process at different levels of resolution. However, mammalian cell biology has been studied primarily by using in vitro models. Cell culture models have been used the most, since it offers several advantages such as, being amenable to both pharmacological and genetic manipulations, reproducibility, and relatively low costs. However, their major limitation is that the architecture and physiology of cells in vitro differ considerably from the in vivo environment. This issue can be overcome by the use of intravital microscopy, which encompasses various optical microscopy techniques aimed at visualizing biological processes in live animals. Recent developments in non-linear optical microscopy resulted in an enormous increase of in vivo studies, which have addressed key biological questions in fields such as neurobiology, immunology and tumor biology. However, the motion artifacts derived from heartbeat and respiration have prevented the imaging of intracellular structures and limited the use of intravital microscopy to the analysis of tissue architecture or single cell behavior. In this respect, the goals of my thesis have been: 1) the development of an experimental system that enables visualizing subcellular organelles in live rodents for extended periods of time, and 2) the investigation of the role of the actin cytoskeleton in endocytosis and exocytosis in live rodents. Here, I describe the establishment of a model for studying endocytosis end exocytosis in the salivary glands of live rats and mice. Moreover, I show that both processes can be imaged in live animals and that their behavior and kinetics differ from what has been reported in in vitro systems. Next, I show that the salivary glands can be genetically and pharmacologically manipulated in situ, thus opening the door for the investigation of the molecular machinery regulating membrane trafficking in live animals. Finally, in the last part of my dissertation, I focus on specific approaches developed to study the kinetics of exocytosis of single secretory granules and discuss how the actin cytoskeleton plays a fundamental role in controlling this process.Doctor of Philosoph

Regulated Exocytosis: Novel Insights from Intravital Microscopy: Exocytosis and Intravital Microscopy

Regulated exocytosis is a fundamental process that every secretory cell uses to deliver molecules to the cell surface and the extracellular space by virtue of membranous carriers. This process has been extensively studied using various approaches such as biochemistry, electro-physiology, and electron microscopy. However, recent developments in time-lapse light microscopy have made possible imaging individual exocytic events hence advancing our understanding of this process at a molecular level. In this review, we focus on intravital microscopy a light microscopy-based approach that enables imaging subcellular structures in live animals, and discuss its recent application to study regulated exocytosis. Intravital microscopy has revealed differences in regulation and modality of regulated exocytosis between in vitro and in vivo model systems, unraveled novel aspects of this process that can be appreciated only in in vivo settings, and provided valuable and novel information on its molecular machinery. In conclusion, we make the case for intravital microscopy being a mature technique that can be used to investigate the molecular machinery of several intracellular events under physiological conditions

Intravital Two-Photon Microscopy for Studying the Uptake and Trafficking of Fluorescently Conjugated Molecules in Live Rodents

Here we describe an experimental system based on intravital two-photon microscopy for studying endocytosis in live animals. The rodent submandibular glands were chosen as model organs since they can be exposed easily, imaged without compromising their function and, furthermore, they are amenable to pharmacological and genetic manipulations. We show that the fibroblasts within the stroma of the glands readily internalize systemically injected molecules such as fluorescently conjugated dextran and bovine serum albumin, providing a robust model to study endocytosis. We dynamically image the trafficking of these probes from the early endosomes to the late endosomes and lysosomes while also visualizing homotypic fusion events between early endosomes. Finally, we demonstrate that pharmacological agents can be delivered specifically to the submandibular salivary glands thus providing a powerful tool to study the molecular machinery regulating endocytosis in a physiological context

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

Actin coating and compression of fused secretory vesicles are essential for surfactant secretion - a role for Rho, formins and myosin II

Secretion of vesicular contents by exocytosis is a fundamental cellular process. Increasing evidence suggests that post-fusion events play an important role in determining the composition and quantity of the secretory output. In particular, regulation of fusion pore dilation and closure is considered a key regulator of the post-fusion phase. However, depending on the nature of the cargo, additional mechanisms might be essential to facilitate effective release. We have recently described that in alveolar type II (ATII) cells, lamellar bodies (LBs), which are secretory vesicles that store lung surfactant, are coated with actin following fusion with the plasma membrane. Surfactant, a lipoprotein complex, does not readily diffuse out of fused LBs following opening and dilation of the fusion pore. Using fluorescence microscopy, atomic force microscopy and biochemical assays, we present evidence that actin coating and subsequent contraction of the actin coat is essential to facilitate surfactant secretion. Latrunculin B prevents actin coating of fused LBs and inhibits surfactant secretion almost completely. Simultaneous imaging of the vesicle membrane and the actin coat revealed that contraction of the actin coat compresses the vesicle following fusion. This leads to active extrusion of vesicle contents. Initial actin coating of fused vesicles is dependent on activation of Rho and formin-dependent actin nucleation. Actin coat contraction is facilitated by myosin II. In summary, our data suggest that fusion pore opening and dilation itself is not sufficient for release of bulky vesicle cargos and that active extrusion mechanisms are required

Real-Time Measurement of F-Actin Remodelling during Exocytosis Using Lifeact-EGFP Transgenic Animals

F-actin remodelling is essential for a wide variety of cell processes. It is important in exocytosis, where F-actin coats fusing exocytic granules. The purpose of these F-actin coats is unknown. They may be important in stabilizing the fused granules, they may play a contractile role and promote expulsion of granule content and finally may be important in endocytosis. To elucidate these functions of F-actin remodelling requires a reliable method to visualize F-actin dynamics in living cells. The recent development of Lifeact-EGFP transgenic animals offers such an opportunity. Here, we studied the characteristics of exocytosis in pancreatic acinar cells obtained from the Lifeact-EGFP transgenic mice. We show that the time-course of agonist-evoked exocytic events and the kinetics of each single exocytic event are the same for wild type and Lifeact-EGFP transgenic animals. We conclude that Lifeact-EGFP animals are a good model to study of exocytosis and reveal that F-actin coating is dependent on the de novo synthesis of F-actin and that development of actin polymerization occurs simultaneously in all regions of the granule. Our insights using the Lifeact-EGFP mice demonstrate that F-actin coating occurs after granule fusion and is a granule-wide event

Actin depolymerisation and crosslinking join forces with myosin II to contract actin coats on fused secretory vesicles

In many secretory cells actin and myosin are specifically recruited to the surface of secretory granules following their fusion with the plasma membrane. Actomyosin-dependent compression of fused granules is essential to promote active extrusion of cargo. However, little is known about molecular mechanisms regulating actin coat formation and contraction. Here, we provide a detailed kinetic analysis of the molecules regulating actin coat contraction on fused lamellar bodies in primary alveolar type II cells. We demonstrate that ROCK1 and myosin light chain kinase 1 (MLCK1, also known as MYLK) translocate to fused lamellar bodies and activate myosin II on actin coats. However, myosin II activity is not sufficient for efficient actin coat contraction. In addition, cofilin-1 and α-actinin translocate to actin coats. ROCK1-dependent regulated actin depolymerisation by cofilin-1 in cooperation with actin crosslinking by α-actinin is essential for complete coat contraction. In summary, our data suggest a complementary role for regulated actin depolymerisation and crosslinking, and myosin II activity, to contract actin coats and drive secretion

The role of myosin 1c and myosin 1b in surfactant exocytosis

Actin and actin-associated proteins have a pivotal effect on regulated exocytosis in secretory cells and influence pre-fusion as well as post-fusion stages of exocytosis. Actin polymerization on secretory granules during the post-fusion phase (formation of an actin coat) is especially important in cells with large secretory vesicles or poorly soluble secretions. Alveolar type II (ATII) cells secrete hydrophobic lipo-protein surfactant, which does not easily diffuse from fused vesicles. Previous work showed that compression of actin coat is necessary for surfactant extrusion. Here, we investigate the role of class 1 myosins as possible linkers between actin and membranes during exocytosis. Live-cell microscopy showed translocation of fluorescently labeled myosin 1b and myosin 1c to the secretory vesicle membrane after fusion. Myosin 1c translocation was dependent on its pleckstrin homology domain. Expression of myosin 1b and myosin 1c constructs influenced vesicle compression rate, whereas only the inhibition of myosin 1c reduced exocytosis. These findings suggest that class 1 myosins participate in several stages of ATII cell exocytosis and link actin coats to the secretory vesicle membrane to influence vesicle compression