Intracellular Membrane Transport in Vascular Endothelial Cells

The main component of blood and lymphatic vessels is the endothelium covering their luminal surface. It plays a significant role in many cardiovascular diseases. Tremendous progress has been made in deciphering of molecular mechanisms involved into intracellular transport. However, molecular machines are mostly characterized in vitro. It is important to adapt this knowledge to the situation existing in tissues and organs. Moreover, contradictions have accumulated within the field related to the function of endothelial cells (ECs) and their trans-endothelial pathways. This has induced necessity for the re-evaluation of several mechanisms related to the function of vascular ECs and intracellular transport and transcytosis there. Here, we analyze available data related to intracellular transport within ECs and re-examine several hypotheses about the role of different mechanisms in transcytosis across ECs. We propose a new classification of vascular endothelium and hypotheses related to the functional role of caveolae and mechanisms of lipid transport through ECs.</p

Activity of the SNARE Protein SNAP29 at the Endoplasmic Reticulum and Golgi Apparatus

Snap29 is a conserved regulator of membrane fusion essential to complete autophagy and to support other cellular processes, including cell division. In humans, inactivating SNAP29 mutations causes CEDNIK syndrome, a rare multi-systemic disorder characterized by congenital neuro-cutaneous alterations. The fibroblasts of CEDNIK patients show alterations of the Golgi apparatus (GA). However, whether and how Snap29 acts at the GA is unclear. Here we investigate SNAP29 function at the GA and endoplasmic reticulum (ER). As part of the elongated structures in proximity to these membrane compartments, a pool of SNAP29 forms a complex with Syntaxin18, or with Syntaxin5, which we find is required to engage SEC22B-loaded vesicles. Consistent with this, in HeLa cells, in neuroepithelial stem cells, and in vivo, decreased SNAP29 activity alters GA architecture and reduces ER to GA trafficking. Our data reveal a new regulatory function of Snap29 in promoting secretory trafficking

Small cargo proteins and large aggregates can traverse the Golgi by a common mechanism without leaving the lumen of cisternae

Procollagen (PC)-I aggregates transit through the Golgi complex without leaving the lumen of Golgi cisternae. Based on this evidence, we have proposed that PC-I is transported across the Golgi stacks by the cisternal maturation process. However, most secretory cargoes are small, freely diffusing proteins, thus raising the issue whether they move by a transport mechanism different than that used by PC-I. To address this question we have developed procedures to compare the transport of a small protein, the G protein of the vesicular stomatitis virus (VSVG), with that of the much larger PC-I aggregates in the same cell. Transport was followed using a combination of video and EM, providing high resolution in time and space. Our results reveal that PC-I aggregates and VSVG move synchronously through the Golgi at indistinguishable rapid rates. Additionally, not only PC-I aggregates (as confirmed by ultrarapid cryofixation), but also VSVG, can traverse the stack without leaving the cisternal lumen and without entering Golgi vesicles in functionally relevant amounts. Our findings indicate that a common mechanism independent of anterograde dissociative carriers is responsible for the traffic of small and large secretory cargo across the Golgi stack

Endocytic reawakening of motility in jammed epithelia.

Dynamics of epithelial monolayers has recently been interpreted in terms of a jamming or rigidity transition. How cells control such phase transitions is, however, unknown. Here we show that RAB5A, a key endocytic protein, is sufficient to induce large-scale, coordinated motility over tens of cells, and ballistic motion in otherwise kinetically arrested monolayers. This is linked to increased traction forces and to the extension of cell protrusions, which align with local velocity. Molecularly, impairing endocytosis, macropinocytosis or increasing fluid efflux abrogates RAB5A-induced collective motility. A simple model based on mechanical junctional tension and an active cell reorientation mechanism for the velocity of self-propelled cells identifies regimes of monolayer dynamics that explain endocytic reawakening of locomotion in terms of a combination of large-scale directed migration and local unjamming. These changes in multicellular dynamics enable collectives to migrate under physical constraints and may be exploited by tumours for interstitial dissemination

The Kiss-and-Run Model of Intra-Golgi Transport

The Golgi apparatus (GA) is the main station along the secretory pathway. Mechanisms of intra-Golgi transport remain unresolved. Three models compete with each other for the right to be defined as the paradigm. The vesicular model cannot explain the following: (1) lipid droplets and aggregates of procollagen that are larger than coatomer I (COPI)-dependent vesicles are transported across the GA; and (2) most anterograde cargoes are depleted in COPI vesicles. The compartment progression/maturation model has the following problems: (1) most Golgi-resident proteins are depleted in COPI vesicles; (2) there are no COPI vesicles for the recycling of the resident proteins in the &lt;em&gt;trans&lt;/em&gt;-most-Golgi cisterna; and (3) different proteins have different rates of intra-Golgi transport. The diffusion model based on permanent inter-cisternal connections cannot explain the existence of lipid, ionic and protein gradients across the Golgi stacks. In contrast, the kiss-and-run model has the potential to explain most of the experimental observations. The kiss-and-run model can be symmetric when fusion and then fission occurs in the same place, and asymmetric when fusion takes place in one location, whereas fission takes place in another. The asymmetric kiss-and-run model resembles the carrier maturation mechanism, and it can be used to explain the transport of large cargo aggregates

Molecular mechanisms responsible for formation of Golgi ribbon

The formation of the Golgi ribbon takes place in protists and metazoans. It is especially prominent in mammalian cells during interphase. Golgi ribbon formation represents an orchestrated sequence of events based not only on different molecular mechanisms but also on discrete cellular functions. Mechanisms responsible for the generation of the Golgi ribbon include Golgi centralization, cis- and transGolgins, molecular machines responsible for the fusion of cargo domains with cisternal rims, and several other less studied factors. Here, we substantiate the hypothesis that cis-Golgins function mostly not as tethering factors, but are responsible for the attachment of the cis-most cisternae to the medial Golgi stacks, whereas transGolgins are responsible for the attachment of the transmost cisterna to the medial Golgi stacks. This hypothesis is tested analyzing predictions derived from it and related to molecular mechanisms responsible for mitotic fragmentation of Golgi stacks

Membrane curvature, trans-membrane area asymmetry, budding, fission and organelle geometry

In biology, the modern scientific fashion is to mostly study proteins. Much less attention is paid to lipids. However, lipids themselves are extremely important for the formation and functioning of cellular membrane organelles. Here, the role of the geometry of the lipid bilayer in regulation of organelle shape is analyzed. It is proposed that during rapid shape transition, the number of lipid heads and their size (i.e., due to the change in lipid head charge) inside lipid leaflets modulates the geometrical properties of organelles, in particular their membrane curvature. Insertion of proteins into a lipid bilayer and the shape of protein trans-membrane domains also affect the trans-membrane asymmetry between surface areas of luminal and cytosol leaflets of the membrane. In the cases where lipid molecules with a specific shape are not predominant, the shape of lipids (cylindrical, conical, or wedge-like) is less important for the regulation of membrane curvature, due to the flexibility of their acyl chains and their high ability to diffuse

Comparison of the Cisterna Maturation-Progression Model with the Kiss-and-Run Model of Intra-Golgi Transport: Role of Cisternal Pores and Cargo Domains

The Golgi complex is the central station of the secretory pathway. Knowledge about the mechanisms of intra-Golgi transport is inconsistent. Here, we compared the explanatory power of the cisterna maturation-progression model and the kiss-and-run model. During intra-Golgi transport, conventional cargoes undergo concentration and form cisternal distensions or distinct membrane domains that contain only one membrane cargo. These domains and distension are separated from the rest of the Golgi cisternae by rows of pores. After the arrival of any membrane cargo or a large cargo aggregate at the Golgi complex, the cis-Golgi SNAREs become enriched within the membrane of cargo-containing domains and then replaced by the trans-Golgi SNAREs. During the passage of these domains, the number of cisternal pores decreases. Restoration of the cisternal pores is COPI-dependent. Our observations are more in line with the kiss-and-run model