Protein dynamics at invadopodia control invasion-migration transitions in melanoma cells.

Cell invasion is a highly complex process that requires the coordination of cell migration and degradation of the extracellular matrix. In melanoma cells, as in many highly invasive cancer cell types these processes are driven by the regulated formation of adhesives structures such as focal adhesions and invasive structures like invadopodia. Structurally, focal adhesion and invadopodia are quite distinct, yet they share many protein constituents. However, quantitative understanding of the interaction of invadopodia with focal adhesion is lacking, and how invadopodia turn-over is associated with invasion-migration transition cycles remains unknown. In this study, we investigated the role of Pyk2, cortactin and Tks5 in invadopodia turnover and their relation with focal adhesions. We found that active Pyk2 and cortactin are localised at both focal adhesions and invadopodia. At invadopodia, localisation of active Pyk2 is correlated with ECM degradation. During invadopodia disassembly, Pyk2 and cortactin but not Tks5 are often relocated at nearby nascent adhesions. We also show that during ECM degradation, cell migration is reduced which is likely related to the sharing of common molecules within the two structures. Finally, we found that the dual FAK/Pyk2 inhibitor PF-431396 inhibits both focal adhesion and invadopodia activities thereby reducing both migration and ECM degradation.

Inhibiting FAK-Paxillin Interaction Reduces Migration and Invadopodia-Mediated Matrix Degradation in Metastatic Melanoma Cells.

The nonreceptor tyrosine kinase FAK is a promising target for solid tumor treatment because it promotes invasion, tumor progression, and drug resistance when overexpressed. Investigating the role of FAK in human melanoma cells, we found that both in situ and metastatic melanoma cells strongly express FAK, where it controls tumor cells' invasiveness by regulating focal adhesion-mediated cell motility. Inhibiting FAK in human metastatic melanoma cells with either siRNA or a small inhibitor targeting the kinase domain impaired migration but led to increased invadopodia formation and extracellular matrix degradation. Using FAK mutated at Y397, we found that this unexpected increase in invadopodia activity is due to the lack of phosphorylation at this residue. To preserve FAK-Src interaction while inhibiting pro-migratory functions of FAK, we found that altering FAK-paxillin interaction, with either FAK mutation in the focal adhesion targeting (FAT) domain or a competitive inhibitor peptide mimicking paxillin LD domains drastically reduces cell migration and matrix degradation by preserving FAK activity in the cytoplasm. In conclusion, our data show that targeting FAK-paxillin interactions could be a potential therapeutic strategy to prevent metastasis formation, and molecules targeting this interface could be alternative to inhibitors of FAK kinase activity which display unexpected effects.

Zinc Fingers in HIV-1 Gag Precursor Are Not Equivalent for gRNA Recruitment at the Plasma Membrane.

The human immunodeficiency virus type 1 Gag precursor specifically selects the unspliced viral genomic RNA (gRNA) from the bulk of cellular and spliced viral RNAs via its nucleocapsid (NC) domain and drives gRNA encapsidation at the plasma membrane (PM). To further identify the determinants governing the intracellular trafficking of Gag-gRNA complexes and their accumulation at the PM, we compared, in living and fixed cells, the interactions between gRNA and wild-type Gag or Gag mutants carrying deletions in NC zinc fingers (ZFs) or a nonmyristoylated version of Gag. Our data showed that the deletion of both ZFs simultaneously or the complete NC domain completely abolished intracytoplasmic Gag-gRNA interactions. Deletion of either ZF delayed the delivery of gRNA to the PM but did not prevent Gag-gRNA interactions in the cytoplasm, indicating that the two ZFs display redundant roles in this respect. However, ZF2 played a more prominent role than ZF1 in the accumulation of the ribonucleoprotein complexes at the PM. Finally, the myristate group, which is mandatory for anchoring the complexes at the PM, was found to be dispensable for the association of Gag with the gRNA in the cytosol.

Zwitterionic Stealth Dye-Loaded Polymer Nanoparticles for Intracellular Imaging.

Intracellular applications of fluorescent nanoparticles (NPs) as probes and labels are currently limited by significant molecular crowding and the high level of complexity encountered inside living cells. The solution is to develop very small, bright, and noninteracting (stealth) NPs. Combining these properties requires implementing the stealth behavior through the thinnest possible hydrophilic shell. Here, we propose a one-step process for preparing ultrasmall and bright stealth NPs based on a zwitterionic (ZI) methacrylate-based copolymer. Dye-loaded polymer NPs are assembled through nanoprecipitation of the copolymer together with the salt of a rhodamine B derivative and a bulky hydrophobic counterion to achieve high particle brightness. We found that 10 mol % ZI groups in the polymer yield NPs of less than 15 nm that are stable in physiological salt conditions and practically resistant to protein adsorption, as suggested by fluorescence correlation spectroscopy. The combination of the very small size with the nonfouling nature of these particles enables spreading of ZI polymer NPs in the whole cytosol after their microinjection into living cells. In addition, single-particle tracking showed up to four times faster diffusion of ZI NPs in the cytosol compared to PEGylated NPs. The obtained dye-loaded ZI polymer NPs open the route to intracellular single-particle tracking and biosensing applications.

FAK regulates dynein localisation and cell polarity in migrating mouse fibroblasts.

BACKGROUND: Fibroblasts executing directional migration position their centrosome, and their Golgi apparatus, in front of the nucleus towards the cell leading edge. Centrosome positioning relative to the nucleus has been associated to mechanical forces exerted on the centrosome by the microtubule-dependent molecular motor cytoplasmic dynein 1, and to nuclear movements such as rearward displacement and rotation events. Dynein has been proposed to regulate the position of the centrosome by exerting pulling forces on microtubules from the cell leading edge, where the motor is enriched during migration. However, the mechanism explaining how dynein acts at the front of the cells has not been elucidated. RESULTS: We present here results showing that the protein Focal Adhesion Kinase (FAK) interacts with dynein and regulates the enrichment of the dynein/dynactin complex at focal adhesions at the cell the leading edge of migrating fibroblasts. This suggests that focal adhesions provide anchoring sites for dynein during the polarisation process. In support of this, we present evidence indicating that the interaction between FAK and dynein, which is regulated by the phosphorylation of FAK on its Ser732 residue, is required for proper centrosome positioning. Our results further show that the polarisation of the centrosome can occur independently of nuclear movements. Although FAK regulates both nuclear and centrosome motilities, downregulating the interaction between FAK and dynein affects only the nuclear independent polarisation of the centrosome. CONCLUSIONS: Our work highlights the role of FAK as a key player in the regulation of several aspects of cell polarity. We thus propose a model in which the transient localisation of dynein with focal adhesions provides a tuneable mechanism to bias dynein traction forces on microtubules allowing proper centrosome positioning in front of the nucleus. SIGNIFICANCE: We unravel here a new role for the cancer therapeutic target FAK in the regulation of cell morphogenesis.

Targeting Focal Adhesion Kinase Using Inhibitors of Protein-Protein Interactions.

Focal adhesion kinase (FAK) is a cytoplasmic non-receptor protein tyrosine kinase that is overexpressed and activated in many human cancers. FAK transmits signals to a wide range of targets through both kinase-dependant and independent mechanism thereby playing essential roles in cell survival, proliferation, migration and invasion. In the past years, small molecules that inhibit FAK kinase function have been developed and show reduced cancer progression and metastasis in several preclinical models. Clinical trials have been conducted and these molecules display limited adverse effect in patients. FAK contain multiple functional domains and thus exhibit both important scaffolding functions. In this review, we describe the major FAK interactions relevant in cancer signalling and discuss how such knowledge provide rational for the development of Protein-Protein Interactions (PPI) inhibitors.

Role of the nucleocapsid domain in HIV-1 Gag oligomerization and trafficking to the plasma membrane: a fluorescence lifetime imaging microscopy investigation.

The Pr55 Gag of human immunodeficiency virus type 1 orchestrates viral particle assembly in producer cells, which requires the genomic RNA and a lipid membrane as scaffolding platforms. The nucleocapsid (NC) domain with its two invariant CCHC zinc fingers flanked by unfolded basic sequences is thought to direct genomic RNA selection, dimerization and packaging during virus assembly. To further investigate the role of NC domain, we analyzed the assembly of Gag with deletions in the NC domain in parallel with that of wild-type Gag using fluorescence lifetime imaging microscopy combined with Förster resonance energy transfer in HeLa cells. We found that, upon binding to nucleic acids, the NC domain promotes the formation of compact Gag oligomers in the cytoplasm. Moreover, the intracellular distribution of the population of oligomers further suggests that oligomers progressively assemble during their trafficking toward the plasma membrane (PM), but with no dramatic changes in their compact arrangement. This ultimately results in the accumulation at the PM of closely packed Gag oligomers that likely arrange in hexameric lattices, as revealed by the perfect match between the experimental Förster resonance energy transfer value and the one calculated from the structural model of Gag in immature viruses. The distal finger and flanking basic sequences, but not the proximal finger, appear to be essential for Gag oligomer compaction and membrane binding. Moreover, the full NC domain was found to be instrumental in the kinetics of Gag oligomerization and intracellular trafficking. These findings further highlight the key roles played by the NC domain in virus assembly.

Fluorescent amino acid undergoing excited state intramolecular proton transfer for site-specific probing and imaging of peptide interactions.

Fluorescent amino acids bearing environment-sensitive fluorophores are highly valuable tools for site-selective probing of peptide/ligand interactions. Herein, we synthesized a fluorescent l-amino acid bearing the 4'-methoxy-3-hydroxyflavone fluorophore (M3HFaa) that shows dual emission, as a result of an excited state intramolecular proton transfer (ESIPT). The dual emission of M3HFaa was found to be substantially more sensitive to hydration as compared to previous analogues. By replacing the Ala30 and Trp37 residues of a HIV-1 nucleocapsid peptide, M3HFaa was observed to preserve the peptide structure and functions. Interaction of the labeled peptides with nucleic acids and lipid vesicles produced a strong switch in their dual emission, favoring the emission of the ESIPT product. This switch was associated with the appearance of long-lived fluorescence lifetimes for the ESIPT product, as a consequence of the rigid environment in the complexes that restricted the relative motions of the M3HFaa aromatic moieties. The strongest restriction and thus the longest fluorescence lifetimes were observed at position 37 in complexes with nucleic acids, where the probe likely stacks with the nucleobases. Based on the dependence of the lifetime values on the nature of the ligand and the labeled position, two-photon fluorescence lifetime imaging was used to identify the binding partners of the labeled peptides microinjected into living cells. Thus, M3HFaa appears as a sensitive tool for monitoring site selectively peptide interactions in solution and living cells.

Altering FAK-paxillin interactions reduces adhesion, migration and invasion processes.

Focal adhesion kinase (FAK) plays an important role in signal transduction pathways initiated at sites of integrin-mediated cell adhesion to the extracellular matrix. Thus, FAK is involved in many aspects of the metastatic process including adhesion, migration and invasion. Recently, several small molecule inhibitors which target FAK catalytic activity have been developed by pharmaceutical companies. The current study was aimed at addressing whether inhibiting FAK targeting to focal adhesions (FA) represents an efficient alternative strategy to inhibit FAK downstream pathways. Using a mutagenesis approach to alter the targeting domain of FAK, we constructed a FAK mutant that fails to bind paxillin. Inhibiting FAK-paxillin interactions led to a complete loss of FAK localization at FAs together with reduced phosphorylation of FAK and FAK targets such as paxillin and p130Cas. This in turn resulted in altered FA dynamics and inhibition of cell adhesion, migration and invasion. Moreover, the migration properties of cells expressing the FAK mutant were reduced as compared to FAK-/- cells. This was correlated with a decrease in both phospho-Src and phospho-p130Cas levels at FAs. We conclude that targeting FAK-paxillin interactions is an efficient strategy to reduce FAK signalling and thus may represent a target for the development of new FAK inhibitors.

Contribution of soft substrates to malignancy and tumor suppression during colon cancer cell division.

In colon cancer, a highly aggressive disease, progression through the malignant sequence is accompanied by increasingly numerous chromosomal rearrangements. To colonize target organs, invasive cells cross several tissues of various elastic moduli. Whether soft tissue increases malignancy or in contrast limits invasive colon cell spreading remains an open question. Using polyelectrolyte multilayer films mimicking microenvironments of various elastic moduli, we revealed that human SW480 colon cancer cells displayed increasing frequency in chromosomal segregation abnormalities when cultured on substrates with decreasing stiffness. Our results show that, although decreasing stiffness correlates with increased cell lethality, a significant proportion of SW480 cancer cells did escape from the very soft substrates, even when bearing abnormal chromosome segregation, achieve mitosis and undergo a new cycle of replication in contrast to human colonic HCoEpiC cells which died on soft substrates. This observation opens the possibility that the ability of cancer cells to overcome defects in chromosome segregation on very soft substrates could contribute to increasing chromosomal rearrangements and tumor cell aggressiveness.

The tumor suppressor Apc controls planar cell polarities central to gut homeostasis.

The stem cells (SCs) at the bottom of intestinal crypts tightly contact niche-supporting cells and fuel the extraordinary tissue renewal of intestinal epithelia. Their fate is regulated stochastically by populational asymmetry, yet whether asymmetrical fate as a mode of SC division is relevant and whether the SC niche contains committed progenitors of the specialized cell types are under debate. We demonstrate spindle alignments and planar cell polarities, which form a novel functional unit that, in SCs, can yield daughter cell anisotropic movement away from niche-supporting cells. We propose that this contributes to SC homeostasis. Importantly, we demonstrate that some SC divisions are asymmetric with respect to cell fate and provide data suggesting that, in some SCs, mNumb displays asymmetric segregation. Some of these processes were altered in apparently normal crypts and microadenomas of mice carrying germline Apc mutations, shedding new light on the first stages of progression toward colorectal cancer.

FAK phosphorylation at Tyr-925 regulates cross-talk between focal adhesion turnover and cell protrusion.

Cell migration is a highly complex process that requires the coordinated formation of membrane protrusion and focal adhesions (FAs). Focal adhesion kinase (FAK), a major signaling component of FAs, is involved in the disassembly process of FAs through phosphorylation and dephosphorylation of its tyrosine residues, but the role of such phosphorylations in nascent FA formation and turnover near the cell front and in cell protrusion is less well understood. In the present study, we demonstrate that, depending on the phosphorylation status of Tyr-925 residue, FAK modulates cell migration via two specific mechanisms. FAK⁻/⁻ mouse embryonic fibroblasts (MEFs) expressing nonphosphorylatable Y925F-FAK show increased interactions between FAK and unphosphorylated paxillin, which lead to FA stabilization and thus decreased FA turnover and reduced cell migration. Conversely, MEFs expressing phosphomimetic Y925E-FAK display unchanged FA disassembly rates, show increase in phosphorylated paxillin in FAs, and exhibit increased formation of nascent FAs at the cell leading edges. Moreover, Y925E-FAK cells present enhanced cell protrusion together with activation of the p130(CAS)/Dock180/Rac1 signaling pathway. Together, our results demonstrate that phosphorylation of FAK at Tyr-925 is required for FAK-mediated cell migration and cell protrusion.

HIV-1 Vpr oligomerization but not that of Gag directs the interaction between Vpr and Gag.

During HIV-1 assembly, the viral protein R (Vpr) is incorporated into newly made viral particles via an interaction with the C-terminal domain of the Gag polyprotein precursor Pr55(Gag). Vpr has been implicated in the nuclear import of newly made viral DNA and subsequently in its transcription. In addition, Vpr can affect the cell physiology by causing G(2)/M cell cycle arrest and apoptosis. Vpr can form oligomers, but their roles have not yet been investigated. We have developed fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer-based assays to monitor the interaction between Pr55(Gag) and Vpr in HeLa cells. To that end, we used enhanced green fluorescent protein-Vpr that can be incorporated into the virus and tetracysteine (TC)-tagged Pr55(Gag)-TC. This TC motif is tethered to the C terminus of Pr55(Gag) and does not interfere with Pr55(Gag) trafficking and the assembly of virus-like particles (VLPs). Results show that the Pr55(Gag)-Vpr complexes accumulated mainly at the plasma membrane. In addition, results with Pr55(Gag)-TC mutants confirm that the (41)LXXLF domain of Gag-p6 is essential for Pr55(Gag)-Vpr interaction. We also report that Vpr oligomerization is crucial for Pr55(Gag) recognition and its accumulation at the plasma membrane. On the other hand, Pr55(Gag)-Vpr complexes are still formed when Pr55(Gag) carries mutations impairing its multimerization. These findings suggest that Pr55(Gag)-Vpr recognition and complex formation occur early during Pr55(Gag) assembly.

Sumoylation delays the ATF7 transcription factor subcellular localization and inhibits its transcriptional activity.

Over the past few years, small ubiquitin-like modifier (SUMO) modification has emerged as an important regulator of diverse pathways and activities including protein localization and transcriptional regulation. We identified a consensus sumoylation motif (IKEE), located within the N-terminal activation domain of the ATF7 transcription factor and thus investigated the role of this modification. ATF7 is a ubiquitously expressed transcription factor, homologous to ATF2, that binds to CRE elements within specific promoters. This protein is able to heterodimerize with Jun or Fos proteins and its transcriptional activity is mediated by interaction with TAF12, a subunit of the general transcription factor TFIID. In the present article, we demonstrate that ATF7 is sumoylated in vitro (using RanBP2 as a E3-specific ligase) and in vivo. Moreover, we show that ATF7 sumoylation affects its intranuclear localization by delaying its entry into the nucleus. Furthermore, SUMO conjugation inhibits ATF7 transactivation activity by (i) impairing its association with TAF12 and (ii) blocking its binding-to-specific sequences within target promoters.

ZW10 function in mitotic checkpoint control, dynein targeting and membrane trafficking: is dynein the unifying theme?

ZW10 was initially identified as a mitotic checkpoint protein involved in chromosome segregation. It was subsequently implicated in targeting cytoplasmic dynein and dynactin to mitotic kinetochores, though the relationship between these functions remains incompletely understood. Recent studies have revealed that ZW10 performs important functions in nondividing cells as well. These include cytoplasmic dynein targeting to Golgi and other membranes, but also SNARE-mediated ER-Golgi trafficking. Identifying a unifying function for ZW10 in these diverse contexts has been elusive, but likely involves cytoplasmic dynein, as discussed here.

Role of the kinetochore/cell cycle checkpoint protein ZW10 in interphase cytoplasmic dynein function.

Zeste white 10 (ZW10) is a mitotic checkpoint protein and the anchor for cytoplasmic dynein at mitotic kinetochores, though it is expressed throughout the cell cycle. We find that ZW10 localizes to pericentriolar membranous structures during interphase and cosediments with Golgi membranes. Dominant-negative ZW10, anti-ZW10 antibody, and ZW10 RNA interference (RNAi) caused Golgi dispersal. ZW10 RNAi also dispersed endosomes and lysosomes. Live imaging of Golgi, endosomal, and lysosomal markers after reduced ZW10 expression showed a specific decrease in the frequency of minus end-directed movements. Golgi membrane-associated dynein was markedly decreased, suggesting a role for ZW10 in dynein cargo binding during interphase. We also find ZW10 enriched at the leading edge of migrating fibroblasts, suggesting that ZW10 serves as a general regulator of dynein function throughout the cell cycle.

A role for cytoplasmic dynein and LIS1 in directed cell movement.

Cytoplasmic dynein has been implicated in numerous aspects of intracellular movement. We recently found dynein inhibitors to interfere with the reorientation of the microtubule cytoskeleton during healing of wounded NIH3T3 cell monolayers. We now find that dynein and its regulators dynactin and LIS1 localize to the leading cell cortex during this process. In the presence of serum, bright diffuse staining was observed in regions of active ruffling. This pattern was abolished by cytochalasin D, and was not observed in cells treated with lysophosphatidic acid, conditions which allow microtubule reorientation but not forward cell movement. Under the same conditions, using total internal reflection fluorescence microscopy, clear punctate dynein/dynactin containing structures were observed along the sides and at the tips of microtubules at the leading edge. Overexpression of dominant negative dynactin and LIS1 cDNAs or injection of antidynein antibody interfered with the rate of cell migration. Together, these results implicate a leading edge cortical pool of dynein in both early and persistent steps in directed cell movement.

Differential regulation of dynein-driven melanosome movement.

Cytoplasmic dyneins are multisubunit minus-end-directed microtubule motors. Different isoforms of dynein are thought to provide a means for independent movement of different organelles. We investigated the differential regulation of dynein-driven transport of pigment organelles (melanosomes) in Xenopus melanophores. Aggregation of melanosomes to the cell center does not change the localization of mitochondria, nor does dispersion of melanosomes cause a change in the perinuclear localization of the Golgi complex, indicating that melanosomes bear a dedicated form of dynein. We examined the subcellular fractionation behavior of dynein light intermediate chains (LIC) and identified at least three forms immunologically, only one of which fractionated with melanosomes. Melanosome aggregation was specifically blocked after injection of an antibody recognizing this LIC. Our data indicate that melanosome-associated dynein is regulated independently of bulk cytoplasmic dynein and involves a subfraction of dynein with a distinct subunit composition.

Function of dynein and dynactin in herpes simplex virus capsid transport.

After fusion of the viral envelope with the plasma membrane, herpes simplex virus type 1 (HSV1) capsids are transported along microtubules (MTs) from the cell periphery to the nucleus. The motor ATPase cytoplasmic dynein and its multisubunit cofactor dynactin mediate most transport processes directed toward the minus-ends of MTs. Immunofluorescence microscopy experiments demonstrated that HSV1 capsids colocalized with cytoplasmic dynein and dynactin. We blocked the function of dynein by overexpressing the dynactin subunit dynamitin, which leads to the disruption of the dynactin complex. We then infected such cells with HSV1 and measured the efficiency of particle binding, virus entry, capsid transport to the nucleus, and the expression of immediate-early viral genes. High concentrations of dynamitin and dynamitin-GFP reduced the number of viral capsids transported to the nucleus. Moreover, viral protein synthesis was inhibited, whereas virus binding to the plasma membrane, its internalization, and the organization of the MT network were not affected. We concluded that incoming HSV1 capsids are propelled along MTs by dynein and that dynein and dynactin are required for efficient viral capsid transport to the nucleus.

LIS1, CLIP-170's key to the dynein/dynactin pathway.

CLIP-170 is a plus-end tracking protein which may act as an anticatastrophe factor. It has been proposed to mediate the association of dynein/dynactin to microtubule (MT) plus ends, and it also binds to kinetochores in a dynein/dynactin-dependent fashion, both via its C-terminal domain. This domain contains two zinc finger motifs (proximal and distal), which are hypothesized to mediate protein-protein interactions. LIS1, a protein implicated in brain development, acts in several processes mediated by the dynein/dynactin pathway by interacting with dynein and other proteins. Here we demonstrate colocalization and direct interaction between CLIP-170 and LIS1. In mammalian cells, LIS1 recruitment to kinetochores is dynein/dynactin dependent, and recruitment there of CLIP-170 is dependent on its site of binding to LIS1, located in the distal zinc finger motif. Overexpression of CLIP-170 results in a zinc finger-dependent localization of a phospho-LIS1 isoform and dynactin to MT bundles, raising the possibility that CLIP-170 and LIS1 regulate dynein/dynactin binding to MTs. This work suggests that LIS1 is a regulated adapter between CLIP-170 and cytoplasmic dynein at sites involved in cargo-MT loading, and/or in the control of MT dynamics.