Daniel G. Jay

The TSC1 tumour suppressor hamartin regulates cell adhesion through ERM proteins and the gtpase Rho

Loss of the tumour-suppressor gene TSC1 is responsible for hamartoma development in tuberous sclerosis complex (TSC), which renders several organs susceptible to benign tumours. Hamartin, the protein encoded by TSC1, contains a coiled-coil domain and is expressed in most adult tissues, although its function is unknown. Here we show that hamartin interacts with the ezrin-radixin-moesin (ERM) family of actin-binding proteins. Inhibition of hamartin function in cells containing focal adhesions results in loss of adhesion to the cell substrate, whereas overexpression of hamartin in cells lacking focal adhesions results in activation of the small GTP-binding protein Rho, assembly of actin stress fibres and formation of focal adhesions. Interaction of endogenous hamartin with ERM-family proteins is required for activation of Rho by serum or by lysophosphatidic acid (LPA). Our data indicate that disruption of adhesion to the cell matrix through loss of hamartin may initiate the development of TSC hamartomas and that a Rho-mediated signalling pathway regulating cell adhesion may constitute a rate-limiting step in tumour formation.

Essential functions of ezrin in maintenance of cell shape and lamellipodial extension in normal and transformed fibroblasts.

BACKGROUND Changes in cell shape and motility are important manifestations of oncogenic transformation, but the mechanisms underlying these changes and key effector molecules in the cytoskeleton remain unknown. The Fos oncogene induces expression of ezrin, the founder member of the ezrin/radixin/moesin (ERM) protein family, but not expression of the related ERM proteins, suggesting that ezrin has a distinct role in cell transformation. ERM proteins have been suggested to link the plasma membrane to the actin-based cytoskeleton and are substrates and anchoring sites for a variety of protein kinases. Here, we examined the role of ezrin in cellular transformation. RESULTS Fos-mediated transformation of Rat-1 fibroblasts resulted in an increased expression and hyperphosphorylation of ezrin, and a concomitant increased association of ezrin with the cortical cytoskeleton. We tagged ezrin with green fluorescent protein and examined its distribution in normal and Fos-transformed fibroblasts: ezrin was concentrated at the leading edge of extending pseudopodia of Fos-transformed Rat-1 cells, and was mainly cytosolic in normal Rat-1 cells. Functional ablation of ezrin by micro-CALI (chromophore-assisted laser inactivation) blocked plasma-membrane ruffling and motility of Fos-transformed fibroblasts. Ablation of ezrin in normal Rat-1 cells caused a marked collapse of the leading edge of the cell. CONCLUSIONS Ezrin plays an important role in pseudopodial extension in Fos-transformed Rat-1 fibroblasts, and maintains cell shape in normal Rat-1 cells. The increased expression, hyperphosphorylation and subcellular redistribution of ezrin upon fibroblast transformation coupled with its roles in cell shape and motility suggest a critical role for ezrin in oncogenic transformation.

Function of myosin-V in filopodial extension of neuronal growth cones.

The molecular mechanisms underlying directed motility of growth cones have not been determined. The role of myosin-V, an unconventional myosin, in growth cone dynamics was examined by chromophore-assisted laser inactivation (CALI). CALI of purified chick brain myosin-V absorbed onto nitrocellulose-coated cover slips inhibited the ability of myosin-V to translocate actin filaments. CALI of myosin-V in growth cones of chick dorsal root ganglion neurons resulted in rapid filopodial retraction. The rate of filopodial extension was significantly decreased, whereas the rate of filopodial retraction was not affected, which suggests a specific role for myosin-V in filopodial extension.

Secretion of extracellular hsp90α via exosomes increases cancer cell motility: a role for plasminogen activation

BackgroundMetastasis is a multi-step process that is responsible for the majority of deaths in cancer patients. Current treatments are not effective in targeting metastasis. The molecular chaperone hsp90α is secreted from invasive cancer cells and activates MMP-2 to enhance invasiveness, required for the first step in metastasis.MethodsWe analyzed the morphology and motility of invasive cancer cells that were treated with exogenous exosomes in the presence or absence of hsp90α. We performed mass spectrometry and immunoprecipitation to identify plasminogen as a potential client protein of extracellular hsp90α. Plasmin activation assays and migration assays were performed to test if plasminogen is activated by extracellular hsp90α and has a role in migration.ResultsWe found that hsp90α is secreted in exosomes in invasive cancer cells and it contributes to their invasive nature. We identified a novel interaction between hsp90α and tissue plasminogen activator that together with annexin II, also found in exosomes, activates plasmin. Extracellular hsp90α promotes plasmin activation as well as increases plasmin dependent cell motility.ConclusionsOur data indicate that hsp90α is released by invasive cancer cells via exosomes and implicates hsp90α in activating plasmin, a second protease that acts in cancer cell invasion.

Extracellular Roles for the Molecular Chaperone, HSP90

Heat shock proteins (hsps) are versatile molecular chaperones that are responsiblefor many cellular functions including proper folding, oligomeric assembly, activation,and transport of proteins. Most of the known roles for hsps involve intracellular proteinsand processes. Mounting evidence suggests that hsps are present and function in theextracellular space. Hsp90 alpha was recently found on the surface and in conditionedmedia of HT-1080 fibrosarcoma cells. Here it acts as a molecular chaperone that assistsin the activation of matrix metalloproteinase-2 (MMP2), leading to increased tumorinvasiveness. Few other extracellular substrates of hsp90 have been identified, butseveral independent observations of extracellular hsp90 suggest that this protein may beimportant for both normal physiology and disease states. Hsp90 typically works in acomplex of associated proteins, and some of these proteins have also been observedextracellularly. Here we show that some of these components, including hsp90organizing protein (hop) and p23, are also found in HT-1080 conditioned mediasupporting the notion that hsp90 complexes function in invasiveness. These findingssuggest a wide-ranging phenomenon of extracellular molecular chaperoning that couldhave implications for biological processes and disease.

Function of a subunit isoforms of the V-ATPase in pH homeostasis and in vitro invasion of MDA-MB231 human breast cancer cells.

It has previously been shown that highly invasive MDA-MB231 human breast cancer cells express vacuolar proton-translocating ATPase (V-ATPases) at the cell surface, whereas the poorly invasive MCF7 cell line does not. Bafilomycin, a specific V-ATPase inhibitor, reduces the in vitro invasion of MB231 cells but not MCF7 cells. Targeting of V-ATPases to different cellular membranes is controlled by isoforms of subunit a. mRNA levels for a subunit isoforms were measured in MB231 and MCF7 cells using quantitative reverse transcription-PCR. The results show that although all four isoforms are detectable in both cell types, levels of a3 and a4 are much higher in MB231 than in MCF7 cells. Isoform-specific small interfering RNAs (siRNA) were employed to selectively reduce mRNA levels for each isoform in MB231 cells. V-ATPase function was assessed using the fluorescent indicators SNARF-1 and pyranine to monitor the pH of the cytosol and endosomal/lysosomal compartments, respectively. Cytosolic pH was decreased only on knockdown of a3, whereas endosome/lysosome pH was increased on knockdown of a1, a2, and a3. Treatment of cells with siRNA to a4 did not affect either cytosolic or endosome/lysosome pH. Measurement of invasion using an in vitro transwell assay revealed that siRNAs to both a3 and a4 significantly inhibited invasion of MB231 cells. Immunofluorescence staining of MB231 cells for V-ATPase distribution revealed extensive intracellular staining, with plasma membrane staining observed in ∼18% of cells. Knockdown of a4 had the greatest effect on plasma membrane staining, leading to a 32% reduction. These results suggest that the a4 isoform may be responsible for targeting V-ATPases to the plasma membrane of MB231 cells and that cell surface V-ATPases play a significant role in invasion. However, other V-ATPases affecting the pH of the cytosol and intracellular compartments, particularly those containing a3, are also involved in invasion.

Myosin 1c and myosin IIB serve opposing roles in lamellipodial dynamics of the neuronal growth cone

The myosin family of motor proteins is implicated in mediating actin-based growth cone motility, but the roles of many myosins remain unclear. We previously implicated myosin 1c (M1c; formerly myosin Iβ) in the retention of lamellipodia (Wang et al., 1996). Here we address the role of myosin II (MII) in chick dorsal root ganglion neuronal growth cone motility and the contribution of M1c and MII to retrograde F-actin flow using chromophore-assisted laser inactivation (CALI). CALI of MII reduced neurite outgrowth and growth cone area by 25%, suggesting a role for MII in lamellipodial expansion. Micro-CALI of MII caused a rapid reduction in local lamellipodial protrusion in growth cones with no effects on filopodial dynamics. This is opposite to micro-CALI of M1c, which caused an increase in lamellipodial protrusion. We used fiduciary beads (Forscher et al., 1992) to observe retrograde F-actin flow during the acute loss of M1c or MII. Micro-CALI of M1c reduced retrograde bead flow by 76%, whereas micro-CALI of MII or the MIIB isoform did not. Thus, M1c and MIIB serve opposite and nonredundant roles in regulating lamellipodial dynamics, and M1c activity is specifically required for retrograde F-actin flow.

CD155/PVR plays a key role in cell motility during tumor cell invasion and migration.

BackgroundInvasion is an important early step of cancer metastasis that is not well understood. Developing therapeutics to limit metastasis requires the identification and validation of candidate proteins necessary for invasion and migration.MethodsWe developed a functional proteomic screen to identify mediators of tumor cell invasion. This screen couples Fluorophore Assisted Light Inactivation (FALI) to a scFv antibody library to systematically inactivate surface proteins expressed by human fibrosarcoma cells followed by a high-throughput assessment of transwell invasion.ResultsUsing this screen, we have identified CD155 (the poliovirus receptor) as a mediator of tumor cell invasion through its role in migration. Knockdown of CD155 by FALI or by RNAi resulted in a significant decrease in transwell migration of HT1080 fibrosarcoma cells towards a serum chemoattractant. CD155 was found to be highly expressed in multiple cancer cell lines and primary tumors including glioblastoma (GBM). Knockdown of CD155 also decreased migration of U87MG GBM cells. CD155 is recruited to the leading edge of migrating cells where it colocalizes with actin and αv-integrin, known mediators of motility and adhesion. Knockdown of CD155 also altered cellular morphology, resulting in cells that were larger and more elongated than controls when plated on a Matrigel substrate.ConclusionThese results implicate a role for CD155 in mediating tumor cell invasion and migration and suggest that CD155 may contribute to tumorigenesis.

Extracellular heat shock protein (Hsp)70 and Hsp90α assist in matrix metalloproteinase-2 activation and breast cancer cell migration and invasion.

Breast cancer is second only to lung cancer in cancer-related deaths in women, and the majority of these deaths are caused by metastases. Obtaining a better understanding of migration and invasion, two early steps in metastasis, is critical for the development of treatments that inhibit breast cancer metastasis. In a functional proteomic screen for proteins required for invasion, extracellular heat shock protein 90 alpha (Hsp90α) was identified and shown to activate matrix metalloproteinase 2 (MMP-2). The mechanism of MMP-2 activation by Hsp90α is unknown. Intracellular Hsp90α commonly functions with a complex of co-chaperones, leading to our hypothesis that Hsp90α functions similarly outside of the cell. In this study, we show that a complex of co-chaperones outside of breast cancer cells assists Hsp90α mediated activation of MMP-2. We demonstrate that the co-chaperones Hsp70, Hop, Hsp40, and p23 are present outside of breast cancer cells and co-immunoprecipitate with Hsp90α in vitro and in breast cancer conditioned media. These co-chaperones also increase the association of Hsp90α and MMP-2 in vitro. This co-chaperone complex enhances Hsp90α-mediated activation of MMP-2 in vitro, while inhibition of Hsp70 in conditioned media reduces this activation and decreases cancer cell migration and invasion. Together, these findings support a model in which MMP-2 activation by an extracellular co-chaperone complex mediated by Hsp90α increases breast cancer cell migration and invasion. Our studies provide insight into a novel pathway for MMP-2 activation and suggest Hsp70 as an additional extracellular target for anti-metastatic drug development.

The clutch hypothesis revisited: ascribing the roles of actin-associated proteins in filopodial protrusion in the nerve growth cone.

We seek to understand how the nerve growth cone acts as a sensory motile machine to respond to chemical cues in the developing embryo. This review focuses on filopodial protrusion and F-actin-based motility because there is good evidence that these processes are required for axon guidance. The clutch hypothesis, which states that filopodial protrusion occurs by actin assembly when an actin filament is fixed with respect to the substrate (i.e., a clutch is engaged), was postulated by Mitchison and Kirscher to link protrusion to actin dynamics. Protrusion would require functional modules for movement of material into filopodia, clutching the F-actin, F-actin assembly at the tip, and retrograde flow. In this review, recent studies of actin-associated proteins involved in filopodial protrusion will be summarized, and their roles will be assessed in the context of the clutch hypothesis. The large number of proteins involved in filopodial motility and their complex interactions make it difficult to understand how these proteins act in protrusion. Recently, we have used microscale chromophore-assisted laser inactivation (micro-CALI) for the focal and acute inactivation of specific actin-associated proteins during filopodial protrusion to address their in situ roles. Our findings suggest that myosin V functions in moving membranes or other material forward in extending filopodia, that talin acts in the clutch module, and that zyxin acts in actin assembly at the tip during filopodial protrusion, perhaps by recruiting Ena/VASP family members to promote actin elongation at this site.