Direct lysosome-based autophagy of lipid droplets in hepatocytes.

Hepatocytes metabolize energy-rich cytoplasmic lipid droplets (LDs) in the lysosome-directed process of autophagy. An organelle-selective form of this process (macrolipophagy) results in the engulfment of LDs within double-membrane delimited structures (autophagosomes) before lysosomal fusion. Whether this is an exclusive autophagic mechanism used by hepatocytes to catabolize LDs is unclear. It is also unknown whether lysosomes alone might be sufficient to mediate LD turnover in the absence of an autophagosomal intermediate. We performed live-cell microscopy of hepatocytes to monitor the dynamic interactions between lysosomes and LDs in real-time. We additionally used a fluorescent variant of the LD-specific protein (PLIN2) that exhibits altered fluorescence in response to LD interactions with the lysosome. We find that mammalian lysosomes and LDs undergo interactions during which proteins and lipids can be transferred from LDs directly into lysosomes. Electron microscopy (EM) of primary hepatocytes or hepatocyte-derived cell lines supports the existence of these interactions. It reveals a dramatic process whereby the lipid contents of the LD can be "extruded" directly into the lysosomal lumen under nutrient-limited conditions. Significantly, these interactions are not affected by perturbations to crucial components of the canonical macroautophagy machinery and can occur in the absence of double-membrane lipoautophagosomes. These findings implicate the existence of an autophagic mechanism used by mammalian cells for the direct transfer of LD components into the lysosome for breakdown. This process further emphasizes the critical role of lysosomes in hepatic LD catabolism and provides insights into the mechanisms underlying lipid homeostasis in the liver.

Maturation of Lipophagic Organelles in Hepatocytes Is Dependent Upon a Rab10/Dynamin-2 Complex.

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.

Development and characterization of cholangioids from normal and diseased human cholangiocytes as an in vitro model to study primary sclerosing cholangitis.

Primary sclerosing cholangitis (PSC) is an incurable, fibroinflammatory biliary disease for which there is no effective pharmacotherapy. We recently reported cholangiocyte senescence as an important phenotype in PSC while others showed that portal macrophages accumulate in PSC. Unfortunately, our ability to explore cholangiocyte senescence and macrophage accumulation has been hampered by limited in vitro models. Thus, our aim was to develop and characterize a three-dimensional (3D) model of normal and diseased bile ducts (cholangioids) starting with normal human cholangiocytes (NHC), senescent NHC (NHC-sen), and cholangiocytes from PSC patients. In 3D culture, NHCs formed spheroids of ~5000 cells with a central lumen of ~150 µm. By confocal microscopy and western blot, cholangioids retained expression of cholangiocyte proteins (cytokeratin 7/19) and markers of epithelial polarity (secretin receptor and GM130). Cholangioids are functionally active, and upon secretin stimulation, luminal size increased by ~80%. Cholangioids exposed to hydrogen peroxide exhibited cellular senescence and the senescence-associated secretory phenotype (SASP; increased IL-6, p21, SA-ß-Gal, yH2A.x and p16 expression). Furthermore, cholangioids derived from NHC-sen or PSC patients were smaller and had slower growth than the controls. When co-cultured with THP-1 macrophages, the number of macrophages associated with NHC-sen or PSC cholangioids was five- to seven-fold greater compared to co-culture with non-senescent NHC. We observed that NHC-sen and PSC cholangioids release greater number of extracellular vesicles (EVs) compared to controls. Moreover, conditioned media from NHC-sen cholangioids resulted in an ~2-fold increase in macrophage migration. In summary, we developed a method to generate normal and diseased cholangioids, characterized them morphologically and functionally, showed that they can be induced to senescence and SASP, and demonstrated both EV release and macrophage attraction. This novel model mimics several features of PSC, and thus will be useful for studying the pathogenesis of PSC and potentially identifying new therapeutic targets.

HBV secretion is regulated through the activation of endocytic and autophagic compartments mediated by Rab7 stimulation.

The cellular mechanisms by which hepatitis B virus (HBV) is assembled and exported are largely undefined. Recently, it has been suggested that these steps require the multivesicular body (MVB) and the autophagic machinery. However, the mechanisms by which HBV might regulate these compartments are unclear. In this study, we have found that by activating Rab7a, HBV alters its own secretion by inducing dramatic changes in the morphology of MVB and autophagic compartments. These changes are characterized by the formation of numerous tubules that are dependent upon the increase in Rab7 activity observed in the HBV-expressing HepG2.2.15 cells compared to HepG2 cells. Interestingly, transfection-based expression of the five individual viral proteins indicated that the precore protein, which is a precursor of HBeAg, was largely responsible for the increased Rab7 activity. Finally, small interfering RNA (siRNA)-mediated depletion of Rab7 significantly increased the secretion of virions, suggesting that reduced delivery of the virus to the lysosome facilitates viral secretion. These findings provide novel evidence indicating that HBV can regulate its own secretion through an activation of the endo-lysosomal and autophagic pathway mediated by Rab7 activation.

Ethanol disrupts hepatocellular lipophagy by altering Rab5-centric LD-lysosome trafficking.

BACKGROUND: Previous reports suggest that lipid droplets (LDs) in the hepatocyte can be catabolized by a direct engulfment from nearby endolysosomes (microlipophagy). Further, it is likely that this process is compromised by chronic ethanol (EtOH) exposure leading to hepatic steatosis. This study investigates the hepatocellular machinery supporting microlipophagy and EtOH-induced alterations in this process with a focus on the small, endosome-associated, GTPase Rab5. METHODS AND RESULTS: Here we report that this small Ras-related GTPase is a resident component of LDs, and its activity is important for hepatocellular LD-lysosome proximity and physical interactions. We find that Rab5 siRNA knockdown causes an accumulation of LDs in hepatocytes by inhibiting lysosome dependent LD catabolism. Importantly, Rab5 appears to support this process by mediating the recruitment of early endosomal and or multivesicular body compartments to the LD surface before lysosome fusion. Interestingly, while wild-type or a constituently active GTPase form (Q79L) of Rab5 supports LD-lysosome transport, this process is markedly reduced in cells expressing a GTPase dead (S34N) Rab5 protein or in hepatocytes exposed to chronic EtOH. CONCLUSIONS: These findings support the novel premise of an early endosomal/multivesicular body intermediate compartment on the LD surface that provides a "docking" site for lysosomal trafficking, not unlike the process that occurs during the hepatocellular degradation of endocytosed ligands that is also known to be compromised by EtOH exposure.

Modeling Gut Neuro-Epithelial Connections in a Novel Micro uidic Device.

Organs that face external environments, such as skin and gut, are lined by epithelia, which have two functions - to provide a semi-permeable barrier and to sense stimuli. The intestinal lumen is filled with diverse chemical and physical stimuli. Intestinal epithelial cells sense these stimuli and signal to enteric neurons which coordinate a range of physiologic processes required for normal digestive tract function. Yet, the neuro-epithelial connections between intestinal epithelial cells and enteric neurons remain poorly resolved, which leaves us with limited mechanistic understanding of their function. We describe the development of a two-compartment microfluidic device for modeling neuro-epithelial interactions, and apply it to form the gut's neuro-epithelial connections. The device contains epithelial and neuronal compartments connected by microgrooves. The epithelial compartment was designed for cell seeding via injection and confinement of intestinal epithelial cells derived from human intestinal organoids. We demonstrated that organoids planarized effectively and retained epithelial phenotype for over a week. In the second chamber we dissociated and cultured intestinal myenteric neurons including intrinsic primary afferent neurons (IPANs) from transgenic mice that expressed the fluorescent protein tdTomato. IPANs extended projections into microgrooves, surrounded and frequently made contacts with epithelial cells. The density and directionality of neuronal projections were enhanced by the presence of epithelial cells in the adjacent compartment. Our microfluidic device represents a platform for dissecting structure and function of neuro-epithelial connections in the gut and other organs (skin, lung, bladder, and others) in health and disease.

Ectopic expression of DOCK8 regulates lysosome-mediated pancreatic tumor cell invasion.

Amplified lysosome activity is a hallmark of pancreatic ductal adenocarcinoma (PDAC) orchestrated by oncogenic KRAS that mediates tumor growth and metastasis, though the mechanisms underlying this phenomenon remain unclear. Using comparative proteomics, we found that oncogenic KRAS significantly enriches levels of the guanine nucleotide exchange factor (GEF) dedicator of cytokinesis 8 (DOCK8) on lysosomes. Surprisingly, DOCK8 is aberrantly expressed in a subset of PDAC, where it promotes cell invasion in vitro and in vivo. DOCK8 associates with lysosomes and regulates lysosomal morphology and motility, with loss of DOCK8 leading to increased lysosome size. DOCK8 promotes actin polymerization at the surface of lysosomes while also increasing the proteolytic activity of the lysosomal protease cathepsin B. Critically, depletion of DOCK8 significantly reduces cathepsin-dependent extracellular matrix degradation and impairs the invasive capacity of PDAC cells. These findings implicate ectopic expression of DOCK8 as a key driver of KRAS-driven lysosomal regulation and invasion in pancreatic cancer cells.

Modeling gut neuro-epithelial connections in a novel microfluidic device.

The intestinal lumen is filled with diverse chemical and physical stimuli. Intestinal epithelial cells sense these stimuli and signal to enteric neurons which coordinate a range of physiologic processes required for normal digestive tract function. Yet, the neuro-epithelial connections remain poorly resolved, in part because the tools for orchestrating interactions between these cellular compartments are lacking. We describe the development of a two-compartment microfluidic device for co-culturing enteric neurons with intestinal epithelial cells. The device contains epithelial and neuronal compartments connected by microgrooves. The epithelial compartment was designed for cell seeding via injection and confinement of intestinal epithelial cells derived from human intestinal organoids. We demonstrated that organoids planarized effectively and retained epithelial phenotype for over a week. In the second chamber we dissociated and cultured intestinal myenteric neurons including intrinsic primary afferent neurons (IPANs) from transgenic mice that expressed the fluorescent protein tdTomato. IPANs extended projections into microgrooves, surrounded and frequently made contacts with epithelial cells. The density and directionality of neuronal projections were enhanced by the presence of epithelial cells in the adjacent compartment. Our microfluidic device represents a platform that may, in the future, be used to dissect structure and function of neuro-epithelial connections in the gut and other organs (skin, lung, bladder, and others) in health and disease.

Hepatocytes internalize trophic receptors at large endocytic "Hot Spots".

UNLABELLED: Clathrin-mediated endocytosis in mammalian epithelial cells is believed to require the synergistic action of structural coat proteins and mechanochemical enzymes to deform and sever the plasma membrane (PM) into discreet vesicles. It is generally believed that the formation of clathrin-coated pits in epithelial cells occurs randomly along the apical and basolateral plasma membranes. In this study we visualized the endocytic machinery in living hepatocytes using green fluorescent protein (GFP)-tagged dynamin, a large mechanochemical guanosine triphosphate (GTP)ase implicated in the liberation of nascent vesicles from the plasma membrane and a variety of internal membrane compartments. Confocal microscopy of living cells expressing the epithelial isoform of GFP-tagged dynamin [Dyn2-GFP] revealed a distribution along the ventral PM in discrete vesicle-like puncta or in large (2-10 µm) tubuloreticular plaques. Remarkably, these large structures are dynamic as they form and then disappear, while generating large numbers of motile endocytic vesicles with which dynamin associates. Inhibiting dynamin function by microinjection of purified dynamin antibodies increases the number and size of the tubuloreticular plaques. Importantly, these "hot spots" sequester specific trophic receptors and cognate ligands such as transferrin receptor 1 (TfR1), but not TfR2. CONCLUSION: These findings suggest that hepatocytes sequester or prerecruit both structural and enzymatic components of the clathrin-based endocytic machinery to functional hot spots, from which large numbers of coated pits form and vesicles are generated. This process may mimic the endocytic organization found at the synapse in neuronal cells.

Anticachectic regulator analysis reveals Perp-dependent antitumorigenic properties of 3-methyladenine in pancreatic cancer.

Approximately 80% of pancreatic cancer patients suffer from cachexia, and one-third die due to cachexia-related complications such as respiratory failure and cardiac arrest. Although there has been considerable research into cachexia mechanisms and interventions, there are, to date, no FDA-approved therapies. A major contributing factor for the lack of therapy options could be the failure of animal models to accurately recapitulate the human condition. In this study, we generated an aged model of pancreatic cancer cachexia to compare cachexia progression in young versus aged tumor-bearing mice. Comparative skeletal muscle transcriptome analyses identified 3-methyladenine (3-MA) as a candidate antiwasting compound. In vitro analyses confirmed antiwasting capacity, while in vivo analysis revealed potent antitumor effects. Transcriptome analyses of 3-MA-treated tumor cells implicated Perp as a 3-MA target gene. We subsequently (a) observed significantly higher expression of Perp in cancer cell lines compared with control cells, (b) noted a survival disadvantage associated with elevated Perp, and (c) found that 3-MA-associated Perp reduction inhibited tumor cell growth. Finally, we have provided in vivo evidence that survival benefits conferred by 3-MA administration are independent of its effect on tumor progression. Taken together, we report a mechanism linking 3-MA to Perp inhibition, and we further implicate Perp as a tumor-promoting factor in pancreatic cancer.

Distinct forms of the actin cross-linking protein α-actinin support macropinosome internalization and trafficking.

The α-actinin family of actin cross-linking proteins have been implicated in driving tumor cell metastasis through regulation of the actin cytoskeleton; however, there has been little investigation into whether these proteins can influence tumor cell growth. We demonstrate that α-actinin 1 and 4 are essential for nutrient uptake through the process of macropinocytosis in pancreatic ductal adenocarcinoma (PDAC) cells, and inhibition of these proteins decreases tumor cell survival in the presence of extracellular protein. The α-actinin proteins play essential roles throughout the macropinocytic process, where α-actinin 4 stabilizes the actin cytoskeleton on the plasma membrane to drive membrane ruffling and macropinosome internalization and α-actinin 1 localizes to actin tails on macropinosomes to facilitate trafficking to the lysosome for degradation. In addition to tumor cell growth, we also observe that the α-actinin proteins can influence uptake of chemotherapeutics and extracellular matrix proteins through macropinocytosis, suggesting that the α-actinin proteins can regulate multiple tumor cell properties through this endocytic process. In summary, these data demonstrate a critical role for the α-actinin isoforms in tumor cell macropinocytosis, thereby affecting the growth and invasive potential of PDAC tumors.

The Exosome-Associated Tetraspanin CD63 Contributes to the Efficient Assembly and Infectivity of the Hepatitis B Virus.

Currently, the hepatocellular trafficking pathways that are used by the hepatitis B virus (HBV) during viral infection and shedding are poorly defined. It is known that the HBV uses late endosomal and multivesicular body (MVB) compartments for assembly and release. The intraluminal vesicles (ILVs) generated within MVBs have also been implicated in the late synthesis stages of a variety of pathogenic viruses. We recently observed that the HBV within infected hepatocytes appears to associate with the tetraspanin protein CD63, known to be a prominent and essential component of ILVs. Immunofluorescence microscopy of HBV-expressing cells showed that CD63 colocalized with HBV proteins (large hepatitis B surface antigens [LHBs] and hepatitis B core) and labeled an exceptionally large number of secreted extracellular vesicles of uniform size. Small interfering RNA (siRNA)-mediated depletion of CD63 induced a substantial accumulation of intracellular LHBs protein but did not alter the levels of either intracellular or extracellular HBV DNA, nor pregenomic RNA. Consistent with these findings, we found that markedly less LHBs protein was associated with the released HBV particles from CD63 siRNA-treated cells. Importantly, the HBV viral particles that were shed from CD63-depleted cells were substantially less infective than those collected from control cells with normal CD63 levels. Conclusion: These findings implicate the tetraspanin protein CD63 as a marker and an important component in the formation and release of infectious HBV particles.

ATM-phosphorylated SPOP contributes to 53BP1 exclusion from chromatin during DNA replication.

53BP1 activates nonhomologous end joining (NHEJ) and inhibits homologous recombination (HR) repair of DNA double-strand breaks (DSBs). Dissociation of 53BP1 from DSBs and consequent activation of HR, a less error-prone pathway than NHEJ, helps maintain genome integrity during DNA replication; however, the underlying mechanisms are not fully understood. Here, we demonstrate that E3 ubiquitin ligase SPOP promotes HR during S phase of the cell cycle by excluding 53BP1 from DSBs. In response to DNA damage, ATM kinase-catalyzed phosphorylation of SPOP causes a conformational change in SPOP, revealed by x-ray crystal structures, that stabilizes its interaction with 53BP1. 53BP1-bound SPOP induces polyubiquitination of 53BP1, eliciting 53BP1 extraction from chromatin by a valosin-containing protein/p97 segregase complex. Our work shows that SPOP facilitates HR repair over NHEJ during DNA replication by contributing to 53BP1 removal from chromatin. Cancer-derived SPOP mutations block SPOP interaction with 53BP1, inducing HR defects and chromosomal instability.

Dynamin 2 interacts with α-actinin 4 to drive tumor cell invasion.

The large GTPase Dynamin 2 (Dyn2) is known to increase the invasiveness of pancreatic cancer tumor cells, but the mechanisms by which Dyn2 regulates changes in the actin cytoskeleton to drive cell migration are still unclear. Here we report that a direct interaction between Dyn2 and the actin-bundling protein alpha-actinin (α-actinin) 4 is critical for tumor cell migration and remodeling of the extracellular matrix in pancreatic ductal adenocarcinoma (PDAC) cells. The direct interaction is mediated through the C-terminal tails of both Dyn2 and α-actinin 4, and these proteins interact at invasive structures at the plasma membrane. While Dyn2 binds directly to both α-actinin 1 and α-actinin 4, only the interaction with α-actinin 4 is required to promote tumor cell invasion. Specific disruption of the Dyn2-α-actinin 4 interaction blocks the ability of PDAC cells to migrate in either two dimensions or invade through extracellular matrix as a result of impaired invadopodia stability. Analysis of human PDAC tumor tissue additionally reveals that elevated α-actinin 4 or Dyn2 expression are predictive of poor survival. Overall, these data demonstrate that Dyn2 regulates cytoskeletal dynamics, in part, by interacting with the actin-binding protein α-actinin 4 during tumor cell invasion.

Pancreatic tumor cell metastasis is restricted by MT1-MMP binding protein MTCBP-1.

The process by which tumor cells mechanically invade through surrounding stroma into peripheral tissues is an essential component of metastatic dissemination. The directed recruitment of the metalloproteinase MT1-MMP to invadopodia plays a critical role in this invasive process. Here, we provide mechanistic insight into MT1-MMP cytoplasmic tail binding protein 1 (MTCBP-1) with respect to invadopodia formation, matrix remodeling, and invasion by pancreatic tumor cells. MTCBP-1 localizes to invadopodia and interacts with MT1-MMP. We find that this interaction displaces MT1-MMP from invadopodia, thereby attenuating their number and function and reducing the capacity of tumor cells to degrade matrix. Further, we observe an inverse correlation between MTCBP-1 and MT1-MMP expression both in cultured cell lines and human pancreatic tumors. Consistently, MTCBP-1-expressing cells show decreased ability to invade in vitro and metastasize in vivo. These findings implicate MTCBP-1 as an inhibitor of the metastatic process.

Lipid droplet size directs lipolysis and lipophagy catabolism in hepatocytes.

Lipid droplet (LD) catabolism in hepatocytes is mediated by a combination of lipolysis and a selective autophagic mechanism called lipophagy, but the relative contributions of these seemingly distinct pathways remain unclear. We find that inhibition of lipolysis, lipophagy, or both resulted in similar overall LD content but dramatic differences in LD morphology. Inhibition of the lipolysis enzyme adipose triglyceride lipase (ATGL) resulted in large cytoplasmic LDs, whereas lysosomal inhibition caused the accumulation of numerous small LDs within the cytoplasm and degradative acidic vesicles. Combined inhibition of ATGL and LAL resulted in large LDs, suggesting that lipolysis targets these LDs upstream of lipophagy. Consistent with this, ATGL was enriched in larger-sized LDs, whereas lipophagic vesicles were restricted to small LDs as revealed by immunofluorescence, electron microscopy, and Western blot of size-separated LDs. These findings provide new evidence indicating a synergistic relationship whereby lipolysis targets larger-sized LDs to produce both size-reduced and nascently synthesized small LDs that are amenable for lipophagic internalization.

A novel endocytic mechanism of epidermal growth factor receptor sequestration and internalization.

Cells form transient, circular dorsal ruffles or "waves" in response to stimulation of receptor tyrosine kinases, including epidermal growth factor receptor (EGFR) or platelet-derived growth factor receptor. These dynamic structures progress inward on the dorsal surface and disappear, occurring concomitantly with a marked reorganization of F-actin. The cellular function of these structures is largely unknown. Here we show that EGF-induced waves selectively sequester and internalize approximately 50% of ligand-bound EGFR from the cell surface. This process requires receptor phosphorylation, active phosphatidylinositol 3-kinase, and dynamin 2, although clathrin-coated pits or caveolae are not required. Epithelial and fibroblast cells stimulated with EGF sequestered EGFR rapidly into waves that subsequently generated numerous receptor-positive tubular-vesicular structures. Electron microscopy confirmed that waves formed along the dorsal membrane surface and extended numerous tubules into the cytoplasm. These findings characterize a structure that selectively sequesters large numbers of activated EGFR for their subsequent internalization, independent of traditional endocytic mechanisms such as clathrin pits or caveolae.

The mitochondrial protein hFis1 regulates mitochondrial fission in mammalian cells through an interaction with the dynamin-like protein DLP1.

The yeast protein Fis1p has been shown to participate in mitochondrial fission mediated by the dynamin-related protein Dnm1p. In mammalian cells, the dynamin-like protein DLP1/Drp1 functions as a mitochondrial fission protein, but the mechanisms by which DLP1/Drp1 and the mitochondrial membrane interact during the fission process are undefined. In this study, we have tested the role of a mammalian homologue of Fis1p, hFis1, and provided new and mechanistic information about the control of mitochondrial fission in mammalian cells. Through differential tagging and deletion experiments, we demonstrate that the intact C-terminal structure of hFis1 is essential for mitochondrial localization, whereas the N-terminal region of hFis1 is necessary for mitochondrial fission. Remarkably, an increased level of cellular hFis1 strongly promotes mitochondrial fission, resulting in an accumulation of fragmented mitochondria. Conversely, cell microinjection of hFis1 antibodies or treatment with hFis1 antisense oligonucleotides induces an elongated and collapsed mitochondrial morphology. Further, fluorescence resonance energy transfer and coimmunoprecipitation studies demonstrate that hFis1 interacts with DLP1. These results suggest that hFis1 participates in mitochondrial fission through an interaction that recruits DLP1 from the cytosol. We propose that hFis1 is a limiting factor in mitochondrial fission and that the number of hFis1 molecules on the mitochondrial surface determines fission frequency.

A dynamin-cortactin-Arp2/3 complex mediates actin reorganization in growth factor-stimulated cells.

The mechanisms by which mammalian cells remodel the actin cytoskeleton in response to motogenic stimuli are complex and a topic of intense study. Dynamin 2 (Dyn2) is a large GTPase that interacts directly with several actin binding proteins, including cortactin. In this study, we demonstrate that Dyn2 and cortactin function to mediate dynamic remodeling of the actin cytoskeleton in response to stimulation with the motogenic growth factor platelet-derived growth factor. On stimulation, Dyn2 and cortactin coassemble into large, circular structures on the dorsal cell surface. These "waves" promote an active reorganization of actin filaments in the anterior cytoplasm and function to disassemble actin stress fibers. Importantly, inhibition of Dyn2 and cortactin function potently blocked the formation of waves and subsequent actin reorganization. These findings demonstrate that cortactin and Dyn2 function together in a supramolecular complex that assembles in response to growth factor stimulation and mediates the remodeling of actin to facilitate lamellipodial protrusion at the leading edge of migrating cells.

Stromal fibroblasts facilitate cancer cell invasion by a novel invadopodia-independent matrix degradation process.

Metastatic invasion of tumors into peripheral tissues is known to rely upon protease-mediated degradation of the surrounding stroma. This remodeling process uses complex, actin-based, specializations of the plasma membrane termed invadopodia that act both to sequester and release matrix metalloproteinases. Here we report that cells of mesenchymal origin, including tumor-associated fibroblasts, degrade substantial amounts of surrounding matrix by a mechanism independent of conventional invadopodia. These degradative sites lack the punctate shape of conventional invadopodia to spread along the cell base and are reticular and/or fibrous in character. In marked contrast to invadopodia, this degradation does not require the action of Src kinase, Cdc42 or Dyn2. Rather, inhibition of Dyn2 causes a marked upregulation of stromal matrix degradation. Further, expression and activity of matrix metalloproteinases are differentially regulated between tumor cells and stromal fibroblasts. This matrix remodeling by fibroblasts increases the invasive capacity of tumor cells, thereby illustrating how the tumor microenvironment can contribute to metastasis. These findings provide evidence for a novel matrix remodeling process conducted by stromal fibroblasts that is substantially more effective than conventional invadopodia, distinct in structural organization and regulated by disparate molecular mechanisms.