Molecular basis of membrane remodeling during secretion at the plasma membrane. Secretory epithelia such as salivary glands (SGs), mammary glands (MGs) and the liver represent three robust model systems to study various aspects of the remodeling of membranes during intracellular trafficking processes, such as constitutive and regulated protein- and lipid-secretion, and plasma membrane homeostasis. 1) Regulated exocytosis in salivary glands In SG acinar cells, secretory proteins are packed in large granules at the trans-Golgi network (TGN) and transported to the cell periphery where they fuse with the APM upon stimulation of GPCRs, thus releasing their content into the acinar canaliculi. Concomitantly, the membranes of the secretory granules gradually integrate into the APM, thus undergoing substantial remodeling.
Our aim i s to elucidate the molecular machinery regulating the integration of the secretory granules with the APM. To this end, we developed an experimental system in live rodents aimed at imaging and tracking individual secretory granules. We established that upon stimulation of the beta-adrenergic receptor, the granules fuse with the APM, followed, after a short delay, by the recruitment of a complex composed of F-actin and two isoforms of non-muscle myosin II (NMIIA and NMIIB). We showed that actomyosin contractile activity regulates the integration of the granular membranes into the APM and the completion of exocytosis. Last year, we focused on elucidating the mechanisms of recruitment and regulation of NMII. We showed that NMIIA and NMIIB are recruited onto the SGs after their fusion with the APM, and that their contractile activity drives the gradual integration of the granules into the APM. This contrasts with other cellular processes where actomyosin-based contractions employ only one isoform of NMII. By using conditional knock-out mice we determined that NMIIB is required to control the initial steps of the integration of the granular membrane, by stabilizing the F-actin scaffold and providing a continuous contractile activity that pushes the membranes towards the APM. On the other hand, NMIIA is required at later stages of the process to control the expansion of the fusion pore. Since both NMIIA and NMIIB are recruited after the formation of the F-actin scaffold, we assumed that this process would be mediated by their well-characterized actin-binding site. Unexpectedly, we found that both NMII isoforms are recruited in an actin-independent fashion and that the main role of F-actin is to facilitate the proper assembly of the NMII filaments. Indeed, F-actin facilitates the recruitment of the myosin light chain kinase (MLCK), which in turn activates both NMII isoforms via the phosphorylation of two residues (S19 and T18) which initiate the formation of contractile filaments. Finally, we discovered that three members of the Septin family of GTPases, Septin 2, 6, and 7, are recruited on the SGs after their fusion with the APM, and control the activation of MLCK. These results provide a springboard to begin investigating the biophysical basis underlying the process of membrane integration. 2) Lipid droplets secretion in mammary glands In MGs, the lipid droplet (LD) fraction of milk supplies preformed lipids for neonatal development, and the assembled LDs are secreted by a unique apocrine mechanism, that has never been investigated in vivo. To this end, we developed a method for the intravital imaging of mammary cells in transgenic mice that express fluorescently tagged marker proteins. For the first time, we described the kinetic analysis of LD growth and secretion at peak lactation in real time. LD transit from basal to apical regions was slow (0-2 um/min) and frequently intermittent. Droplets grew by the fusion of preexisting droplets, with no restriction on the size of fusogenic partners. Most droplet expansion took several hours and occurred in APM nucleation centers, either close to or in association with the apical surface. Interestingly, droplets were coated with F-actin and NMIIA, although their function is, at the moment, poorly understood. Large droplets gradually pushed the APM, inducing its extensive deformation, which resulted in the budding of the droplets into the apical lumen. Droplets continued to expand as they were emerging from the cell. Contrary to expectations, LDs attached to the APM, but still associated with the cytoplasm were released after oxytocin-mediated contraction of the myoepithelium. This initial investigation will serve as a basis to unravel the machinery regulating the deformation and the budding of the apical plasma membrane. Plasma membrane homeostasis in the liver The bile canaliculi network in the liver is formed by the apical domains of the hepatocytes. Its homeostasis is the result of a balance between endo- and exocytic processes, which tightly regulate the flux of membranes and the maintenance of functional tight junctions.
Our aim i s to investigate the mechanisms that regulate the homeostasis of the bile canaliculi and ensure their proper functionality. As a first step, we determined 1) a high-resolution structure of the bile network in vivo, by using a combination of high-resolution confocal and serial block-face scanning electron microscopy; and 2) its dynamic behavior by monitoring changes in the canaliculi structure and in bile transport, by using time-lapse intravital microscopy. Interestingly, we observed that bile canaliculi undergo continuous peristaltic contractions and expansions, which facilitate bile secretion. We found that this process is mediated by the actomyosin cytoskeleton, as inhibition of F-actin assembly or a RhoKinase controlling NMIIA dramatically blocks the peristaltic movements and delays biliary flow. In addition, we investigated the role of the liver kinase B1 (LKB1) and its downstream effector AMP-activated protein kinase (AMPK), which play critical roles in polarity establishment by regulating membrane trafficking and energy metabolism. To this end, we used liver-specific (albumin-Cre) LKB1 knockout mice (LKB1(-/-). We found that LKB1 plays a fundamental role in the maintenance of functional tight junction (TJ) in vivo. Transmission electron microscopy examination revealed that hepatocyte apical membrane with microvilli substantially extended into the basolateral domain of LKB1(-/-) livers. Immunofluorescence studies revealed that loss of LKB1 led to longer and wider canalicular structures correlating with mislocalization of the junctional protein, cingulin. To test junctional function, we used intravital microscopy to quantify the transport kinetics of 6-carboxyfluorescein diacetate (6-CFDA), which is processed in hepatocytes into its fluorescent derivative 6-carboxyfluorescein (6-CF) and secreted into the canaliculi. In LKB1(-/-) mice, 6-CF remained largely in hepatocytes, canalicular secretion was delayed, and 6-CF appeared in the blood. To test whether 6-CF was transported through permeable TJ, we intravenously injected low molecular weight (3 kDa) dextran in combination with 6-CFDA. In wild-type mice, 3 kDa dextran remained in the vasculature, whereas it rapidly appeared in the abnormal bile canaliculi in LKB1(-/-) mice, confirming that junctional disruption resulted in paracellular exchange between the blood stream and the bile canaliculus. These studies have unraveled a key role for both the actomyosin cytoskeleton and molecules implicated in membrane trafficking and cell metabolism.
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