Autophagosomes are double-membrane vesicles generated during autophagy. Biogenesis of the autophagosome requires membrane acquisition from intracellular compartments, the mechanisms of which are unclear. We previously found that a relocation of COPII machinery to the ER-Golgi intermediate compartment (ERGIC) generates ERGIC-derived COPII vesicles which serve as a membrane precursor for the lipidation of LC3, a key membrane component of the autophagosome. Here we employed super-resolution microscopy to show that starvation induces the enlargement of ER-exit sites (ERES) positive for the COPII activator, SEC12, and the remodeled ERES patches along the ERGIC. A SEC12 binding protein, CTAGE5, is required for the enlargement of ERES, SEC12 relocation to the ERGIC, and modulates autophagosome biogenesis. Moreover, FIP200, a subunit of the ULK protein kinase complex, facilitates the starvation-induced enlargement of ERES independent of the other subunits of this complex and associates via its C-terminal domain with SEC12. Our data indicate a pathway wherein FIP200 and CTAGE5 facilitate starvation-induced remodeling of the ERES, a prerequisite for the production of COPII vesicles budded from the ERGIC that contribute to autophagosome formation. Running title: Endomembrane remodeling for autophagy

Clathrin-mediated endocytosis (CME) robustness under elevated membrane tension is maintained by actin assembly-mediated force generation. However, whether more actin assembles at endocytic sites in response to increased load has not previously been investigated. Here actin network ultrastructure at CME sites was examined under low and high membrane tension. Actin and N-WASP spatial organization indicate that actin polymerization initiates at the base of clathrin-coated pits and that the network then grows away from the plasma membrane. Actin network height at individual CME sites was not coupled to coat shape, raising the possibility that local differences in mechanical load feed back on assembly. By manipulating membrane tension and Arp2/3 complex activity we tested the hypothesis that actin assembly at CME sites increases in response to elevated load. Indeed, in response to elevated membrane tension, actin grew higher, resulting in greater coverage of the clathrin coat, and CME slowed. When membrane tension was elevated and the Arp2/3 complex was inhibited, shallow clathrin-coated pits accumulated, indicating that this adaptive mechanism is especially crucial for coat curvature generation. We propose that actin assembly increases in response to increased load to ensure CME robustness over a range of plasma membrane tensions. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text]

Microtentacles (McTNs) are thin projections found on circulating tumor cells (CTCs) that can be tens of micrometers long. They have been recognized as a potentially critical factor in metastasis due to their involvement in attachment of CTCs to secondary sites of metastasis. So far little structural characterization has been reported, other than that these projections are based on microtubules (MTs), unlike most other cell projections that are based on actin. Work published to date strongly suggests that stabilization of McTNs increases the risk of metastasis through enhanced reattachment of CTCs [1][2][3]. Stabilization occurs by a number of factors that often increase during progression of cancer, including increased expression of proteins such as vimentin and tau [2,4]. Of particular clinical importance, Taxol stabilizes MTs and as a result stabilizes McTNs. Since Taxol is widely used in treatment of cancer, it is very important to understand the magnitude and structural basis for any increased risk of metastasis. In addition, developing a structural understanding for these effects would help to identify novel drug targets that could compensate for any added risk that treatment with drugs such as Taxol poses. With the goals of understanding McTN structure and the mechanisms by which stabilization of McTNs promote reattachment, we have used cryo-electron microscopy and tomography, scanning electron microscopy (SEM) and stochastic optical reconstruction microscopy (STORM) to characterize the structure and activity of these protrusions.SEM was used to follow the development of McTNs after drug treatment of MDA-MB-231 breast cancer cells with latrunculin (LA) and Taxol, with results shown in Fig. 1. A time course series showed formation of McTN within 3 minutes of drug addition (data not shown), and at 15 minutes (Figure 1b) McTNs longer than 10µm can be seen. We have studied their molecular composition by fluorescence labeling. New advances in super-resolution microscopy have allowed for lateral resolution of 10nm and axial resolution of 20nm, which is ideal for investigating the arrangement of 10nm intermediate filaments (IFs) and 25nm MTs inside McTNs. Using dual-objective STORM we imaged MTs and vimentin IFs in suspended MDA-MB-231 breast cancer cells treated with LA and Taxol. In general, the filaments inside the McTNs are arranged parallel to each other but appear spatially separated (Fig. 2).Resin embedding and sectioning were used to provide a view of structures within thicker parts of the McTNs near the cell body. MDA-MB-231 cells treated with LA and Taxol show structural details including the IFs, MTs and ribosomes (Fig. 3). The separation between MTs and IFs inside the McTN suggests that another protein may be linking these networks. Immunofluorescence experiments revealed the presence of plectin, a cytoskeletal linker protein, inside McTNs (data not shown).Cryo tomography has been used to obtain clear 3D maps of the MT distribution within thin parts of the McTNs (Fig. 4). So far we have seen no IFs in ...

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