Branched Actin Maintains Acetylated Microtubule Network in the Early Secretory Pathway

In the early secretory pathway, the delivery of anterograde cargoes from the endoplasmic reticulum (ER) exit sites (ERES) to the Golgi apparatus is a multi-step transport process occurring via the ER-Golgi intermediate compartment (IC, also called ERGIC). While the role microtubules in ER-to-Golgi transport has been well established, how the actin cytoskeleton contributes to this process remains poorly understood. Here, we report that Arp2/3 inhibition affects the network of acetylated microtubules around the Golgi and induces the accumulation of unusually long RAB1/GM130-positive carriers around the centrosome. These long carriers are less prone to reach the Golgi apparatus, and arrival of anterograde cargoes to the Golgi is decreased upon Arp2/3 inhibition. Our data suggest that Arp2/3-dependent actin polymerization maintains a stable network of acetylated microtubules, which ensures efficient cargo trafficking at the late stage of ER to Golgi transport.

Mechanism of shaping membrane nanostructures of endoplasmic reticulum

Recent advances in super-resolution microscopy revealed the previously unknown nanoscopic level of organization of endoplasmic reticulum (ER), one of the most vital intracellular organelles. Membrane nanostructures of 10- to 100-nm intrinsic length scales, which include ER tubular matrices, ER sheet nanoholes, internal membranes of ER exit sites (ERES), and ER transport intermediates, were discovered and imaged in considerable detail, but the physical factors determining their unique geometrical features remained unknown. Here, we proposed and computationally substantiated a common concept for mechanisms of all ER nanostructures based on the membrane intrinsic curvature as a primary factor shaping the membrane and ultra-low membrane tensions as modulators of the membrane configurations. We computationally revealed a common structural motif underlying most of the nanostructures. We predicted the existence of a discrete series of equilibrium configurations of ER tubular matrices and recovered the one corresponding to the observations and favored by ultra-low tensions. We modeled the nanohole formation as resulting from a spontaneous collapse of elements of the ER tubular network adjacent to the ER sheet edge and calculated the nanohole dimensions. We proposed the ERES membrane to have a shape of a super flexible membrane bead chain, which acquires random walk configurations unless an ultra-low tension converts it into a straight conformation of a transport intermediate. The adequacy of the proposed concept is supported by a close qualitative and quantitative similarity between the predicted and observed configurations of all four ER nanostructures.

Intestinal SEC16B modulates obesity by controlling dietary lipid absorption

ABSTRACTGenome-wide association studies (GWAS) have identified genetic variants in SEC16 homolog B (SEC16B) locus to be associated with obesity and body mass index (BMI) in various populations. SEC16B encodes a scaffold protein located at endoplasmic reticulum (ER) exit sites that is implicated to participate in the trafficking of COPII vesicles in mammalian cells. However, the function of SEC16B in vivo, especially in lipid metabolism, has not been investigated. Here we demonstrated that intestinal SEC16B is required for dietary lipid absorption in mice. We showed that Sec16b intestinal knockout (IKO) mice, especially female mice, were protected from HFD-induced obesity. Loss of SEC16B in intestine dramatically reduced postprandial serum triglyceride output upon intragastric lipid load or during overnight fasting and high-fat diet (HFD) refeeding. Further studies showed that intestinal SEC16B deficiency impaired apoB lipidation and chylomicron secretion. These results revealed that SEC16B plays important roles in dietary lipid absorption, which may shed light on the association between variants in SEC16B and obesity in human.

Identification of two pathways mediating protein targeting from ER to lipid droplets

Pathways localizing proteins to their sites of action within a cell are essential for eukaryotic cell organization and function. Although mechanisms of protein targeting to many organelles have been defined, little is known about how proteins, such as key metabolic enzymes, target from the ER to cellular lipid droplets (LDs). Here, we identify two distinct pathways for ER-to-LD (ERTOLD) protein targeting: early ERTOLD, occurring during LD formation, and late ERTOLD, targeting mature LDs after their formation. By using systematic, unbiased approaches, we identified specific membrane-fusion machinery, including regulators, a tether, and SNARE proteins, that are required for late ERTOLD targeting. Components of this fusion machinery localize to LD-ER interfaces and appear to be organized at ER exit sites (ERES) to generate ER-LD membrane bridges. We also identified multiple cargoes for early and late ERTOLD. Collectively, our data provide a new model for how proteins target LDs from the ER.

An in vitro vesicle formation assay reveals cargo clients and factors that mediate vesicular trafficking

The fidelity of protein transport in the secretory pathway relies on the accurate sorting of proteins to their correct destinations. To deepen our understanding of the underlying molecular mechanisms, it is important to develop a robust approach to systematically reveal cargo proteins that depend on specific sorting machinery to be enriched into transport vesicles. Here, we used an in vitro assay that reconstitutes packaging of human cargo proteins into vesicles to quantify cargo capture. Quantitative mass spectrometry (MS) analyses of the isolated vesicles revealed cytosolic proteins that are associated with vesicle membranes in a GTP-dependent manner. We found that two of them, FAM84B (also known as LRAT domain containing 2 or LRATD2) and PRRC1, contain proline-rich domains and regulate anterograde trafficking. Further analyses revealed that PRRC1 is recruited to endoplasmic reticulum (ER) exit sites, interacts with the inner COPII coat, and its absence increases membrane association of COPII. In addition, we uncovered cargo proteins that depend on GTP hydrolysis to be captured into vesicles. Comparing control cells with cells depleted of the cargo receptors, SURF4 or ERGIC53, we revealed specific clients of each of these two export adaptors. Our results indicate that the vesicle formation assay in combination with quantitative MS analysis is a robust and powerful tool to uncover novel factors that mediate vesicular trafficking and to uncover cargo clients of specific cellular factors.

Mutant Cyclin F Impedes COPII Vesicle-Mediated ER-Golgi Trafficking and ER-Associated Degradation, Inducing ER Stress and Golgi Fragmentation in ALS/FTD

BackgroundMutations in the CCNF gene encoding cyclin F are associated with sporadic and familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia, but the underlying pathophysiological mechanisms are unknown. Proper functioning of the endoplasmic reticulum (ER) is essential for physiological cellular function. MethodsWe used human neuroblastoma SH-SY5Y and human embryonic kidney HEK293T cell lines and mouse primary neurons-overexpressing two familial ALS cyclin F mutants to examine whether mutant ALS/FTD-associated cyclin F perturbs key functions of the ER and Golgi compartments. Specific cellular assays were used to examine ER-Golgi transport (VSVGts045), the budding of vesicles from ER membranes and ER-associated degradation (ERAD). Immunocytochemistry was used to examine the morphology of the Golgi and ER-exit sites, and to detect ER stress and apoptosis. Western blotting was used to examine the content of vesicles budding from ER membranes and the interaction between Sec 31 and cyclin F. Flow cytometry was used to examine cell death.Results We demonstrated that mutant cyclin F inhibited protein transport from the ER to Golgi apparatus by a mechanism involving aberrant vesicle sorting from the ER. It also impeded ER-associated degradation, whereby misfolded ER proteins are ubiquitinated and degraded by the proteasome. This was associated with induction of ER stress and Golgi fragmentation, leading to apoptosis. Conclusion Together, these results demonstrate that ER dysfunction is a pathogenic pathway associated with ALS/FTD-variant cyclin F.

Activation of salt Inducible Kinases, IRE1 and PERK leads to Sec bodies formation in Drosophila S2 cells

The phase separation of the non-membrane bound Sec bodies occurs in Drosophila S2 cells by coalescence of components of the ER exit sites under the stress of amino-acid starvation. Here we address which signaling pathways cause Sec body formation and find that two pathways are critical. The first is the activation of the salt inducible kinases (SIK) by Na+ stress, that when it is strong is sufficient. The second is activation of IRE1 and PERK downstream of ER stress induced by absence of amino- acids, which needs to be combined with moderate salt stress to induce Sec body formation. SIK and IRE1/PERK activation appear to potentiate each other through the stimulation of the unfolded protein response, a key parameter in Sec body formation. This work pioneers the role of SIK in phase transition and re-enforces the role of IRE1 and PERK as a metabolic sensor for the level of circulating amino-acids and salt.

COPII collar defines the boundary between ER and ER exit site and does not coat cargo containers

COPII and COPI mediate the formation of membrane vesicles translocating in opposite directions within the secretory pathway. Live-cell and electron microscopy revealed a novel mode of function for COPII during cargo export from the ER. COPII is recruited to membranes defining the boundary between the ER and ER exit sites, facilitating selective cargo concentration. Using direct observation of living cells, we monitored cargo selection processes, accumulation, and fission of COPII-free ERES membranes. CRISPR/Cas12a tagging, the RUSH system, and pharmaceutical and genetic perturbations of ER-Golgi transport demonstrated that the COPII coat remains bound to the ER–ERES boundary during protein export. Manipulation of the cargo-binding domain in COPII Sec24B prohibits cargo accumulation in ERES. These findings suggest a role for COPII in selecting and concentrating exported cargo rather than coating Golgi-bound carriers. These findings transform our understanding of coat proteins’ role in ER-to-Golgi transport.

Aspergillus nidulans biofilm formation modifies cellular architecture and enables light-activated autophagy

After growing on surfaces, including those of medical and industrial importance, fungal biofilms self-generate internal microenvironments. We previously reported that gaseous microenvironments around founder Aspergillus nidulans cells change during biofilm formation causing microtubules (MTs) to disassemble under control of the hypoxic transcription factor SrbA. Here we investigate if biofilm formation might also promote changes to structures involved in exocytosis and endocytosis. During biofilm formation the ER remained intact but ER exit sites and the Golgi apparatus were modified as were endocytic actin patches. The biofilm driven changes required the SrbA hypoxic transcription factor and could be triggered by nitric oxide, further implicating gaseous regulation of biofilm cellular architecture. By tracking GFP-Atg8 dynamics, biofilm founder cells were also observed to undergo autophagy. Most notably, biofilm cells that had undergone autophagy were triggered into further autophagy by spinning disc confocal light. Our findings indicate that fungal biofilm formation modifies the secretory and endocytic apparatus and show biofilm cells can also undergo autophagy that is reactivated by light. The findings provide new insights into the changes occurring in fungal biofilm cell biology that potentially impact their unique characteristics, including antifungal drug resistance. [Media: see text]