Chen, Jing

ABSTRACT Epithelial cells, such as liver-resident hepatocytes, rely heavily on the Rab family of small GTPases to perform membrane trafficking events that dictate cell physiology and metabolism. Not surprisingly, disruption of several Rab proteins can manifest in metabolic diseases or cancer. Rab32 is expressed in many secretory epithelial cells but its role in cellular metabolism is virtually unknown. In this study, we find that Rab32 associates with lysosomes and regulates proliferation and cell size of Hep3B hepatoma and HeLa cells. Specifically, we identify that Rab32 supports the mechanistic target of rapamycin complex 1 (mTORC1) signaling under basal and amino acid-stimulated conditions. Consistent with inhibited mTORC1, an increase in nuclear TFEB localization and lysosome biogenesis is also observed in Rab32-depleted cells. Finally, we find that Rab32 interacts with mTOR kinase, and that loss of Rab32 reduces the association of mTOR and mTORC1 pathway proteins with lysosomes, suggesting that Rab32 regulates lysosomal mTOR trafficking. In summary, these findings suggest that Rab32 functions as a novel regulator of cellular metabolism through supporting mTORC1 signaling. This article has an associated First Person interview with the first author of the paper.

Background and Aims Hepatocytes play a central role in storage and utilization of fat by the liver. Selective breakdown of lipid droplets (LDs) by autophagy (also called lipophagy) is a key process utilized to catabolize these lipids as an energy source. How the autophagic machinery is selectively targeted to LDs, where it mediates membrane engulfment and subsequent degradation, is unclear. Recently, we have reported that two distinct GTPases, the mechanoenzyme, dynamin2 (Dyn2), and the small regulatory Rab GTPase, Rab10, work independently at distinct steps of lipophagy in hepatocytes. Approach and Results In an attempt to understand how these proteins are regulated and recruited to autophagic organelles, we performed a nonbiased biochemical screen for Dyn2‐binding partners and found that Dyn2 actually binds Rab10 directly through a defined effector domain of Rab10 and the middle domain of Dyn2. These two GTPases can be observed to interact transiently on membrane tubules in hepatoma cells and along LD‐centric autophagic membranes. Most important, we found that a targeted disruption of this interaction leads to an inability of cells to trim tubulated cytoplasmic membranes, some of which extend from lipophagic organelles, resulting in LD accumulation. Conclusions This study identifies a functional, and direct, interaction between Dyn2 and a regulatory Rab GTPase that may play an important role in hepatocellular metabolism.

Abstract Metastatic invasion of tumors into peripheral tissues is known to rely upon protease-mediated degradation of the surrounding stroma. This remodeling process is dependent upon complex, actin-based specializations of the plasma membrane termed “invadopodia” that act to both sequester and release matrix metalloproteases. Here we report that cells of mesenchymal origin, including fibroblasts, pancreatic stellate cells, and tumor-associated fibroblasts, degrade substantial amounts of surrounding matrix by a mechanism independent of conventional invadopodia. First, as tumor cell-derived invadopodia are dependent upon the action of the GTPase Dyn2, loss of Dyn2 function inhibits invadopodia-based matrix degradation by pancreatic tumor cells. In surprising contrast, we have found that loss of Dyn2 function in mesenchymal cells does not inhibit degradation, but actually leads to a marked increase. Second, these degradative sites lack the punctate shape of conventional invadopodia but are instead spread along the cell base and are reticular and/or fibrous in character. Third, inhibition of either Src or Cdc42 function, known to drive invadopodia formation, had no effect on the mesenchymal matrix degradation induced by loss of Dyn2. Taken together, these data indicate that this mechanism of degradation is independent of classical invadopodial degradation. To define the mechanistic processes that differ between tumor and stromal cell types, we screened for matrix metalloprotease activity using zymography. An unexpected and significant upregulation of MMP2 activity in mesenchymal-derived cells upon loss of Dyn2 was observed, whereas MMP2 was nearly completely absent in the tumor cells. Further, loss of Dyn2 resulted in an upregulation of surface-bound MT1-MMP, known to activate MMP2. In contrast, loss of Dyn2 in the pancreatic tumor cells reduced surface levels of MT1-MMP. Thus, in mesenchymal cells, loss of Dyn2 induces retention of MT1-MMP at the surface, leading to large scale matrix degradation through an MT1-MMP/MMP2-dependent mechanism. Excitingly, co-culture invasion experiments demonstrate that the matrix degradation induced in these stromal fibroblasts is sufficient to promote invasion by pancreatic tumor cells. These findings provide evidence for a novel matrix remodeling process conducted by mesenchymal cells that is substantially more effective than conventional invadopodia, distinct in structural organization, independent of the classical invadopodial machinery, and capable of promoting invasion by pancreatic tumor cells. Supported by NCI RO1 CA104125 to MAM. Citation Format: Hong Cao, Robbin Eppinga, Eugene Krueger, Jing Chen, Gina Razidlo, Mark A. McNiven. Differential mechanisms utilized by pancreatic tumor cells and associated fibroblasts to remodel stroma during metastatic invasion. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3162. doi:10.1158/1538-7445.AM2014-3162

Immature dendrites extend many actin-rich filopodial structures that can be replaced by synapse-containing dendritic spines as the neuron matures. The large GTPase dynamin-3 (Dyn3) is a component of the postsynapse in hippocampal neurons but its function is undefined. Here, we demonstrate that a specific Dyn3 variant (Dyn3baa) promotes the formation of immature dendritic filopodia in cultured neurons. This effect is dependent upon Dyn3 GTPase activity and a direct interaction with the F-actin-binding protein cortactin. Consistent with these findings, Dyn3baa binds to cortactin with a 200% higher affinity than Dyn3aaa, a near identical isoform that does not induce dendritic filopodia when expressed in cultured neurons. Finally, levels of Dyn3baa-encoding mRNA are tightly regulated during neuronal maturation and are markedly upregulated during synaptogenesis. Together, these findings provide the first evidence that an enhanced interaction between a specific Dyn3 splice variant and cortactin modulate actin-membrane dynamics in developing neurons to regulate the morphogenesis of dendritic spines.