04_polarity. The formation of synaptic vesicles

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1 Brefeldin prevents assembly of the coats required for budding Nocodazole disrupts microtubules Constitutive: coatomer-coated Selected: clathrin-coated The formation of synaptic vesicles Nerve cells (and some endocrine cells) Dense core secretory vesicles (standard) Tiny secretory vesicles Synaptic vesicles that store neurotransmitters (acetylcholine, glutamate, GABA) for rapid signaling at chemical synapsis. 58

2 Precursors of lysosomal hydrolases are covalently modified by addition of mannose-6-phosphate in the cis Golgi network. They interact with M6P receptors and are segregated in the trans Golgi network in clathrin-coated vesicles. At the low ph of the late endosome the hydrolases dissociate from the receptors, which are recycled. The M6P is removed from the hydrolyses to prevent return to the Golgi apparatus with the receptors. 59

3 Non polarized cells Polarized cells 60

4 Segregation is thought to occur in the trans-golgi network (TGN) and is mediated by sorting signals embedded within the protein structure. Apical sorting signals include N- and O-linked glycans in the ectodomain, glycosyl phosphatidylinositol and transmembrane anchors, and amino-acid stretches in the cytoplasmic domain. In contrast, basolateral sorting signals usually comprise amino acid stretches in the cytoplasmic domain that typically include tyrosine, dileucine and monoleucine motifs, as well as clusters of acidic amino acids. Early ideas of how epithelial cells generate and maintain c e l l - s u r f a c e polarity: Kreitzer et al. NATURE CELL BIOLOGY VOL 5 FEBRUARY 2003 CAN SORTING AND DELIVERY OF MEMBRANE PROTEINS FROM THE TGN EXPLAIN THE ESTABLISHMENT OF CELL-SURFACE POLARITY? Two groups have shown that apical and basolateral membrane proteins are sorted at the level of the TGN into separate transport vesicles in fibroblasts. However, these proteins had a non-polarized distribution in the plasma membrane, demonstrating that sorting is not sufficient to generate cell-surface polarity. Drubin and Nelson: Origins of cell polarity. Cell 84: 335,

5 Requirement of spatial cues at the surface for biogenesis of epithelial cell polarity Intracellular sorting and delivery are secondary events 62

6 To examine directly the dynamics of E-cadherin in living cells, we constructed a fusion protein composed of fulllength canine E-cadherin fused at the carboxyl terminus to GFP (EcadGFP). EcadGFP was expressed in MDCK cells, HEK 293 EBNA cells and L cells. plasmide E Cadherin cdna GFP Transfection (lipofectamin) Expression of the Fusion protein 63

7 Hek-293 A: apical mb B: basal mb MDCK EcadGFP properties are similar to those of endogenous E- cadherin: it binds catenins, is targeted to cell-cell contacts, promotes adhesion. L-cells after 18h of aggregation in suspension culture in the presence or absence of extracellular Ca++. Parental L-cells do not express E-cadherin EcadGFP plaques 64

8 Redistribution of EcadGFP during cell-cell contact occurs in two stages and correlates with reorganization of the actin cytoskeleton. <1 hour >2 hours Actin (rhodamine phalloidin) EcadGFP 65

9 Double immunohistochemistry on formaldehyde fixed cells Rhodamine falloidin E-cadherin mab early EcadGFP puncta were associated with thin cables of actin filaments that emerged from circumferential actin cables oriented parallel to the contact. A diffuse pool of E-cadherin clusters into puncta in response to cell cell contact and those puncta organize and become stabilized around actin filaments located close to the contacting membranes. late EcadGFP plaques were sites at which circumferential actin cables terminated. The gradual increase in the amount of EcadGFP in these plaques could be the result of de novo clustering of EcadGFP around new actin filaments exposed at the margins of the cell cell contact, or from the aggregation and migration of puncta that had preformed along the length of the contact. To distinguish between these two possibilities, time-lapse images of EcadGFP plaques were recorded. Quantitative fluorescence intensities of EcadGFP. The average (gray circles) and maximum (black diamonds) intensities in a 20-mm2 region surrounding a developing plaque area are plotted over a 40-min period. Thus, plaques most likely to arise by lateral clustering of a subset of EcadGFP puncta already formed along the cell cell contact, and are perhaps supplemented by recruitment of additional EcadGFP molecules in the area of plaque formation. It is clear that the peak density(black diamonds), but not the total amount (gray circles) of EcadGFP increased in the region of the membrane containing the forming plaque. 66

10 To gain further insight into the assembly dynamics of E-cadherin puncta and plaques, we developed a photobleaching recovery method to measure the diffusion coefficient, mobile fraction, and redistribution of EcadGFP during different stages of contact development Rhodamine falloidin Anti-E-cadherin Ecad-GFP fluorescence of the nonbleached region (red) and the entire cell (green) was monitored during recovery. Images taken before, 0.1 min after, and 10 min after photobleaching a 5.8-mm-diameter circle in a region of membrane not involved in cell cell contact (Membrane), a region of membrane in a, 15-min-old contact (New contact), a region of membrane in the middle of a,60-min-old contact (Puncta), and a membrane at the edge of a.2-h-old contact (Plaque). For each experiment, 300 images were collected every 3.2 s at 0.11 mm/pixel. The relative fluorescence is scaled to the pre-bleach intensity value. The mobile fraction of EcadGFP in the new contact (blue), puncta (green), and plaque (orange) is 100, 50, and,10%, respectively. The circles mark the photobleach region, and the colors correspond to the recovery curves. 67

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12 Factors involved in vesicle delivery from the mother cell into the bud 69

13 S: supernatant P: pellet 70

14 Immunoblotting of postnuclear supernatants from contact-naive and confluent monolayers of MDCK cells showed that similar amounts of Sec6 and Sec8 were present in both non-polarized and polarized cells (Figure 2A), demonstrating that expression of the Sec6/8 complex was not induced by cell cell adhesion. To determine whether the subcellular distribution of the Sec6/8 complex changed during development of epithelial polarity, the fraction of the complex associated with plasma membrane was quantified at different times following initiation of cell cell contacts. E-cadherin, was confined to the top two fractions in Opti- Prep gradients in contact-naive and polarized MDCK cells (Figure 2B). In contact-naive cells,.90% of Sec6 and Sec8 migrated to a position near the bottom of the gradient, indicating that the Sec6/8 complex was largely present in a cytosolic pool. In contrast, Opti-Prep separation of polarized MDCK cell extracts showed that z70% of Sec6 and Sec8 migrated near the top of the gradient, in the same fractions that contained the plasma membrane protein E-cadherin. Therefore, in fully polarized MDCK cell monolayers, the Sec6/8 complex is primarily associated with the plasma membrane. 71

15 The kinetics of recruitment of the Sec6/8 complex to the plasma membrane were assessed by measuring the amounts of Sec6 and Sec8 recovered in the top two fractions from Opti-Prep gradients of homogenates of MDCK cells that had formed cell cell contacts for 3, 6, 12, or 24 hr. This analysis revealed that the Sec6/8 complex is rapidly recruited to the plasma membrane following the onset of cell cell contact with a t1/2» 3 6hr (Figure 2C). Colocalization of Sec6 with E-cadherin and ZO-1 at early cell-cell contacts tightjunction associated protein Sec6 E-cadherin Sec6 ZO-1 methanol Triton X-100 Sec6/8 Complex Associates with Adhesion Protein Complexes at Sites of Early Cell Cell Contacts 72

16 While E-cadherin was broadly distributed along the length of the lateral membrane, both Sec6 and Sec8 were restricted to the apex of the lateral membrane in a distribution similar to that of the tight junction associated protein ZO-1. In budding yeast, the Sec6/8 protein complex has a highly polarized distribution at the bud tip during bud formation (TerBush and Novick, 1995). In polarized MDCKcells, Sec6 and Sec8 were distributed in a circumferential ring around each cell in association with the lateral plasma membrane domain (Figure 4). ZO-1 Anti-sec6 Figure 5. Disruption of Calcium-Dependent Cell Cell Contacts Causes Dissociation of the Sec6/8 Complex from the Plasma Membrane. Polarized MDCK cells cultured on polycarbonate filters were incubated in DMEM containing 1.8 mm calcium (A B) or 5 mm calcium plus 2mM EGTA (C D) for 4 hr at 378C. In (E) (H), cells were cultured in DMEM containing 5 mm calcium plus 2 mm EGTA for 4 hr at 378C, then shifted into medium containing 1.8 mm calcium and incubated for an additional 2.5 hr (E and F) or 7.5 hr (G and H) at 378C. Parallel cultures were fixed, permeabilized, and incubated with antibodies to either ZO-1 (A, C, E, and G) or Sec6 (B, D, F, and H). Antibodies were visualized with fluorescein-labeled donkey anti-rat or anti-membrane, mouse antibodies. control EGTA EGTA then Ca ++ 2,5hr EGTA then Ca ++ 7,5hr 73

17 apical basolateral To synchronize vesicle transport between the Golgi complex and plasma membrane, MDCK cells were infected with recombinant adenoviruses that encode either an apical membrane protein (p75ntr), or a basal-lateral membrane protein (LDL receptor; LDLR). Cells were incubated at 208C for 120 min in the presence of 35S-sulfate to accumulate and label newly synthesized membrane proteins simultaneously in a late Golgi compartment. Cells were permeabilized with streptolysin-o (SLO) and incubated under different conditions. Generalized scheme for the formation of a new membrane domain following induction (cue) of the recruitment of the Sec6/8 complex to the membrane 74