No abstract

Autophagosomes have been reported to form in the vicinity of the endoplasmic reticulum (ER). In many cases, the phagophore membrane is observed between two cisternae of rough ER, but it is not known whether these two membranes are directly connected. To investigate the relationship of the phagophore membrane and the ER, we used electron microscopic tomography of serum and amino acid starved normal rat kidney cells. The cells were fixed in glutaraldehyde and reduced osmium tetroxide and embedded in Epon. Dual axis tilt image series were acquired from two successive 250-nm sections. To analyze the three-dimensional (3D) morphology of phagophores and the associated rough ER, 3D tomograms were used to model the ER and phagophore membranes. The tomographic reconstructions revealed connections between the phagophore/autophagosome membrane and the closely located ER cisternae, especially with the ER located inside the autophagosome. The connections were typically formed by narrow extensions from the phagophore/autophagosome to the ER. This finding has potential implications on the origin of autophagosome membranes, and on the mechanism of phagophore membrane extension. In addition, we observed lipid droplets in very close contact with the phagophores/autophagosomes.

The actin cytoskeleton plays a fundamental role in various motile and morphogenetic processes involving membrane dynamics. We show that actin-binding proteins MIM (missing-in-metastasis) and IRSp53 directly bind PI(4,5)P2-rich membranes and deform them into tubular structures. This activity resides in the N-terminal IRSp53/MIM domain (IMD) of these proteins, which is structurally related to membrane-tubulating BAR (Bin/amphiphysin/Rvs) domains. We found that because of a difference in the geometry of the PI(4,5)P2-binding site, IMDs induce a membrane curvature opposite that of BAR domains and deform membranes by binding to the interior of the tubule. This explains why IMD proteins induce plasma membrane protrusions rather than invaginations. We also provide evidence that the membrane-deforming activity of IMDs, instead of the previously proposed F-actin–bundling or GTPase-binding activities, is critical for the induction of the filopodia/microspikes in cultured mammalian cells. Together, these data reveal that interplay between actin dynamics and a novel membrane-deformation activity promotes cell motility and morphogenesis.

Plant cells are connected through plasmodesmata (PD), membrane-lined channels that allow symplastic movement of molecules between cells. However, little is known about the role of PD-mediated signaling during plant morphogenesis. Here, we describe an Arabidopsis gene, CALS3/GSL12. Gain-of-function mutations in CALS3 result in increased accumulation of callose (β-1,3-glucan) at the PD, a decrease in PD aperture, defects in root development, and reduced intercellular trafficking. Enhancement of CALS3 expression during phloem development suppressed loss-of-function mutations in the phloem abundant callose synthase, CALS7 indicating that CALS3 is a bona fide callose synthase. CALS3 alleles allowed us to spatially and temporally control the PD aperture between plant tissues. Using this tool, we are able to show that movement of the transcription factor SHORT-ROOT and microRNA165 between the stele and the endodermis is PD dependent. Taken together, we conclude that regulated callose biosynthesis at PD is essential for cell signaling.

Highlights d Stretch triggers amplitude-dependent supracellular and nuclear mechanoresponses d H3K9me3 heterochromatin mediates nuclear stiffness and membrane tension d Nuclear deformation-triggered Ca 2+ alters chromatin rheology to prevent DNA damage d Supracellular alignment redistributes stress to restore chromatin state