Mechanical Stabilization of the Glandular Acinus by Linker of Nucleoskeleton and Cytoskeleton Complex.

The nucleoskeleton and cytoskeleton are important protein networks that govern cellular behavior and are connected together by the linker of nucleoskeleton and cytoskeleton (LINC) complex. Mutations in LINC complex components may be relevant to cancer, but how cell-level changes might translate into tissue-level malignancy is unclear. We used glandular epithelial cells in a three-dimensional culture model to investigate the effect of perturbations of the LINC complex on higher order cellular architecture. We show that inducible LINC complex disruption in human mammary epithelial MCF-10A cells and canine kidney epithelial MDCK II cells mechanically destabilizes the acinus. Lumenal collapse occurs because the acinus is unstable to increased mechanical tension that is caused by upregulation of Rho-kinase-dependent non-muscle myosin II motor activity. These findings provide a potential mechanistic explanation for how disruption of LINC complex may contribute to a loss of tissue structure in glandular epithelia.

Imbalanced nucleocytoskeletal connections create common polarity defects in progeria and physiological aging.

Studies of the accelerated aging disorder Hutchinson-Gilford progeria syndrome (HGPS) can potentially reveal cellular defects associated with physiological aging. HGPS results from expression and abnormal nuclear envelope association of a farnesylated, truncated variant of prelamin A called "progerin." We surveyed the diffusional mobilities of nuclear membrane proteins to identify proximal effects of progerin expression. The mobilities of three proteins-SUN2, nesprin-2G, and emerin-were reduced in fibroblasts from children with HGPS compared with those in normal fibroblasts. These proteins function together in nuclear movement and centrosome orientation in fibroblasts polarizing for migration. Both processes were impaired in fibroblasts from children with HGPS and in NIH 3T3 fibroblasts expressing progerin, but were restored by inhibiting protein farnesylation. Progerin affected both the coupling of the nucleus to actin cables and the oriented flow of the cables necessary for nuclear movement and centrosome orientation. Progerin overexpression increased levels of SUN1, which couples the nucleus to microtubules through nesprin-2G and dynein, and microtubule association with the nucleus. Reducing microtubule-nuclear connections through SUN1 depletion or dynein inhibition rescued the polarity defects. Nuclear movement and centrosome orientation were also defective in fibroblasts from normal individuals over 60 y, and both defects were rescued by reducing the increased level of SUN1 in these cells or inhibiting dynein. Our results identify imbalanced nuclear engagement of the cytoskeleton (microtubules: high; actin filaments: low) as the basis for intrinsic cell polarity defects in HGPS and physiological aging and suggest that rebalancing the connections can ameliorate the defects.

TorsinA controls TAN line assembly and the retrograde flow of dorsal perinuclear actin cables during rearward nuclear movement.

The nucleus is positioned toward the rear of most migratory cells. In fibroblasts and myoblasts polarizing for migration, retrograde actin flow moves the nucleus rearward, resulting in the orientation of the centrosome in the direction of migration. In this study, we report that the nuclear envelope-localized AAA+ (ATPase associated with various cellular activities) torsinA (TA) and its activator, the inner nuclear membrane protein lamina-associated polypeptide 1 (LAP1), are required for rearward nuclear movement during centrosome orientation in migrating fibroblasts. Both TA and LAP1 contributed to the assembly of transmembrane actin-associated nuclear (TAN) lines, which couple the nucleus to dorsal perinuclear actin cables undergoing retrograde flow. In addition, TA localized to TAN lines and was necessary for the proper mobility of EGFP-mini-nesprin-2G, a functional TAN line reporter construct, within the nuclear envelope. Furthermore, TA and LAP1 were indispensable for the retrograde flow of dorsal perinuclear actin cables, supporting the recently proposed function for the nucleus in spatially organizing actin flow and cytoplasmic polarity. Collectively, these results identify TA as a key regulator of actin-dependent rearward nuclear movement during centrosome orientation.

Fascin Regulates Nuclear Movement and Deformation in Migrating Cells.

Fascin is an F-actin-bundling protein shown to stabilize filopodia and regulate adhesion dynamics in migrating cells, and its expression is correlated with poor prognosis and increased metastatic potential in a number of cancers. Here, we identified the nuclear envelope protein nesprin-2 as a binding partner for fascin in a range of cell types in vitro and in vivo. Nesprin-2 interacts with fascin through a direct, F-actin-independent interaction, and this binding is distinct and separable from a role for fascin within filopodia at the cell periphery. Moreover, disrupting the interaction between fascin and nesprin-2 C-terminal domain leads to specific defects in F-actin coupling to the nuclear envelope, nuclear movement, and the ability of cells to deform their nucleus to invade through confined spaces. Together, our results uncover a role for fascin that operates independently of filopodia assembly to promote efficient cell migration and invasion.

A mutation abolishing the ZMPSTE24 cleavage site in prelamin A causes a progeroid disorder.

In 1994 in the Journal of Cell Science, Hennekes and Nigg reported that changing valine to arginine at the endoproteolytic cleavage site in chicken prelamin A abolishes its conversion to lamin A. The consequences of this mutation in an organism have remained unknown. We now report that the corresponding mutation in a human subject leads to accumulation of prelamin A and causes a progeroid disorder. Next generation sequencing of the subject and her parents' exomes identified a de novo mutation in the lamin A/C gene (LMNA) that resulted in a leucine to arginine amino acid substitution at residue 647 in prelamin A. The subject's fibroblasts accumulated prelamin A, a farnesylated protein, which led to an increased percentage of cultured cells with morphologically abnormal nuclei. Treatment with a protein farnesyltransferase inhibitor improved abnormal nuclear morphology. This case demonstrates that accumulation of prelamin A, independent of the loss of function of ZMPSTE24 metallopeptidase that catalyzes processing of prelamin A, can cause a progeroid disorder and that a cell biology assay could be used in precision medicine to identify a potential therapy.

FAK, talin and PIPKIγ regulate endocytosed integrin activation to polarize focal adhesion assembly.

Integrin endocytic recycling is critical for cell migration, yet how recycled integrins assemble into new adhesions is unclear. By synchronizing endocytic disassembly of focal adhesions (FAs), we find that recycled integrins reassemble FAs coincident with their return to the cell surface and dependent on Rab5 and Rab11. Unexpectedly, endocytosed integrins remained in an active but unliganded state in endosomes. FAK and Src kinases co-localized with endocytosed integrin and were critical for FA reassembly by regulating integrin activation and recycling, respectively. FAK sustained the active integrin conformation by maintaining talin association with Rab11 endosomes in a type I phosphatidylinositol phosphate kinase (PIPKIγ)-dependent manner. In migrating cells, endocytosed integrins reassembled FAs polarized towards the leading edge, and this polarization required FAK. These studies identify unanticipated roles for FA proteins in maintaining endocytosed integrin in an active conformation. We propose that the conformational memory of endocytosed integrin enhances polarized reassembly of FAs to enable directional cell migration.

Nesprin-2G, a Component of the Nuclear LINC Complex, Is Subject to Myosin-Dependent Tension.

The nucleus of a cell has long been considered to be subject to mechanical force. Despite the observation that mechanical forces affect nuclear geometry and movement, how forces are applied onto the nucleus is not well understood. The nuclear LINC (linker of nucleoskeleton and cytoskeleton) complex has been hypothesized to be the critical structure that mediates the transfer of mechanical forces from the cytoskeleton onto the nucleus. Previously used techniques for studying nuclear forces have been unable to resolve forces across individual proteins, making it difficult to clearly establish if the LINC complex experiences mechanical load. To directly measure forces across the LINC complex, we generated a fluorescence resonance energy transfer-based tension biosensor for nesprin-2G, a key structural protein in the LINC complex, which physically links this complex to the actin cytoskeleton. Using this sensor we show that nesprin-2G is subject to mechanical tension in adherent fibroblasts, with highest levels of force on the apical and equatorial planes of the nucleus. We also show that the forces across nesprin-2G are dependent on actomyosin contractility and cell elongation. Additionally, nesprin-2G tension is reduced in fibroblasts from Hutchinson-Gilford progeria syndrome patients. This report provides the first, to our knowledge, direct evidence that nesprin-2G, and by extension the LINC complex, is subject to mechanical force. We also present evidence that nesprin-2G localization to the nuclear membrane is altered under high-force conditions. Because forces across the LINC complex are altered by a variety of different conditions, mechanical forces across the LINC complex, as well as the nucleus in general, may represent an important mechanism for mediating mechanotransduction.

Moving Cell Boundaries Drive Nuclear Shaping during Cell Spreading.

The nucleus has a smooth, regular appearance in normal cells, and its shape is greatly altered in human pathologies. Yet, how the cell establishes nuclear shape is not well understood. We imaged the dynamics of nuclear shaping in NIH3T3 fibroblasts. Nuclei translated toward the substratum and began flattening during the early stages of cell spreading. Initially, nuclear height and width correlated with the degree of cell spreading, but over time, reached steady-state values even as the cell continued to spread. Actomyosin activity, actomyosin bundles, microtubules, and intermediate filaments, as well as the LINC complex, were all dispensable for nuclear flattening as long as the cell could spread. Inhibition of actin polymerization as well as myosin light chain kinase with the drug ML7 limited both the initial spreading of cells and flattening of nuclei, and for well-spread cells, inhibition of myosin-II ATPase with the drug blebbistatin decreased cell spreading with associated nuclear rounding. Together, these results show that cell spreading is necessary and sufficient to drive nuclear flattening under a wide range of conditions, including in the presence or absence of myosin activity. To explain this observation, we propose a computational model for nuclear and cell mechanics that shows how frictional transmission of stress from the moving cell boundaries to the nuclear surface shapes the nucleus during early cell spreading. Our results point to a surprisingly simple mechanical system in cells for establishing nuclear shapes.

Plus-end tracking proteins, CLASPs, and a viral Akt mimic regulate herpesvirus-induced stable microtubule formation and virus spread.

Although microtubules (MTs) frequently form highly dynamic networks, subsets of MTs become stabilized in response to environmental cues and function as specialized tracks for vesicle and macromolecular trafficking. MT stabilization is controlled by specialized plus-end tracking proteins (+TIPs) whose accumulation at the MT ends is facilitated by the end-binding protein, EB1, and regulated by various signaling pathways. As cargoes themselves, viruses are dependent on MTs for their intracellular movement. Although many viruses affect MT organization, the potential contribution of MT stabilization by +TIPs to infection remains unknown. Here we show that early in infection of primary human fibroblasts, herpes simplex virus type 1 (HSV-1) disrupts the centrosome, the primary MT organizing center in many cell types. As infection progresses HSV-1 induces the formation of stable MT subsets through inactivation of glycogen synthase kinase 3beta by the viral Ser/Thr kinase, Us3. Stable MT formation is reduced in cells infected with Us3 mutants and those stable MTs that form cluster around the trans-Golgi network. Downstream of glycogen synthase kinase 3beta, cytoplasmic linker-associated proteins (CLASPs), specialized host +TIPs that control MT formation at the trans-Golgi network and cortical capture, are specifically required for virus-induced MT stabilization and HSV-1 spread. Our findings demonstrate the biological importance of +TIPs to viral infection and suggest that HSV-1 has evolved to exploit the trans-Golgi network as an alternate MT organizing center to facilitate virus spread.

INF2 promotes the formation of detyrosinated microtubules necessary for centrosome reorientation in T cells.

T cell antigen receptor-proximal signaling components, Rho-family GTPases, and formin proteins DIA1 and FMNL1 have been implicated in centrosome reorientation to the immunological synapse of T lymphocytes. However, the role of these molecules in the reorientation process is not yet defined. Here we find that a subset of microtubules became rapidly stabilized and that their α-tubulin subunit posttranslationally detyrosinated after engagement of the T cell receptor. Formation of stabilized, detyrosinated microtubules required the formin INF2, which was also found to be essential for centrosome reorientation, but it occurred independently of T cell receptor-induced massive tyrosine phosphorylation. The FH2 domain, which was mapped as the INF2 region involved in centrosome repositioning, was able to mediate the formation of stable, detyrosinated microtubules and to restore centrosome translocation in DIA1-, FMNL1-, Rac1-, and Cdc42-deficient cells. Further experiments indicated that microtubule stabilization was required for centrosome polarization. Our work identifies INF2 and stable, detyrosinated microtubules as central players in centrosome reorientation in T cells.

Aquaporin 2 promotes cell migration and epithelial morphogenesis.

The aquaporin 2 (AQP2) water channel, expressed in kidney collecting ducts, contributes critically to water homeostasis in mammals. Animals lacking or having significantly reduced levels of AQP2, however, have not only urinary concentrating abnormalities but also renal tubular defects that lead to neonatal mortality from renal failure. Here, we show that AQP2 is not only a water channel but also an integrin-binding membrane protein that promotes cell migration and epithelial morphogenesis. AQP2 expression modulates the trafficking and internalization of integrin ß1, facilitating its turnover at focal adhesions. In vitro, disturbing the interaction between AQP2 and integrin ß1 by mutating the RGD motif led to reduced endocytosis, retention of integrin ß1 at the cell surface, and defective cell migration and tubulogenesis. Similarly, in vivo, AQP2-null mice exhibited significant retention of integrin ß1 at the basolateral membrane and had tubular abnormalities. In summary, these data suggest that the water channel AQP2 interacts with integrins to promote renal epithelial cell migration, contributing to the structural and functional integrity of the mammalian kidney.

Adenomatous polyposis coli protein nucleates actin assembly and synergizes with the formin mDia1.

The tumor suppressor protein adenomatous polyposis coli (APC) regulates cell protrusion and cell migration, processes that require the coordinated regulation of actin and microtubule dynamics. APC localizes in vivo to microtubule plus ends and actin-rich cortical protrusions, and has well-documented direct effects on microtubule dynamics. However, its potential effects on actin dynamics have remained elusive. Here, we show that the C-terminal "basic" domain of APC (APC-B) potently nucleates the formation of actin filaments in vitro and stimulates actin assembly in cells. Nucleation is achieved by a mechanism involving APC-B dimerization and recruitment of multiple actin monomers. Further, APC-B nucleation activity is synergistic with its in vivo binding partner, the formin mDia1. Together, APC-B and mDia1 overcome a dual cellular barrier to actin assembly imposed by profilin and capping protein. These observations define a new function for APC and support an emerging view of collaboration between distinct actin assembly-promoting factors with complementary activities.

Par3 and dynein associate to regulate local microtubule dynamics and centrosome orientation during migration.

BACKGROUND: Centrosome orientation toward the leading edge of migrating cells depends on dynein and microtubules (MTs), as well as a number of signaling factors at the leading edge. However, centrosomes are maintained at the cell center during orientation in fibroblasts, suggesting that factors working at sites other than the leading edge may also be involved. RESULTS: In a search for factors that function with dynein in centrosome orientation, we found that the polarity protein Par3 associated with dynein and that knockdown of Par3 inhibited centrosome orientation by disrupting the position of the centrosome at the cell center; this disrupted centrosome positioning is the same phenotype as that observed with dynein inhibition. Par3 associated with dynein through its N-terminal dimerization and PDZ1 domains and interacted specifically with dynein light intermediate chain 2 (LIC2). siRNA knockdown of LIC2, but not LIC1, or overexpression of LIC2 or the N-terminal domain of Par3, also inhibited centrosome orientation by disrupting centrosome position. In wound-edge fibroblasts, Par3 specifically localized to cell-cell contacts where it overlapped with MT ends and dynein puncta in a LIC2-dependent fashion. Live imaging showed that MTs exhibited increased pausing at cell-cell contacts compared to the leading edge and that this elevated pausing was dependent on Par3 and LIC2. CONCLUSIONS: Par3 associates with dynein and contributes to the local regulation of MT dynamics at cell-cell contacts and proper positioning of the centrosome at the cell center. We propose that Par3 acts as a cortical factor that tethers MTs through its association with LIC2 dynein.

The formin mDia2 stabilizes microtubules independently of its actin nucleation activity.

A critical microtubule (MT) polarization event in cell migration is the Rho/mDia-dependent stabilization of a subset of MTs oriented toward the direction of migration. Although mDia nucleates actin filaments, it is unclear whether this or a separate activity of mDia underlies MT stabilization. We generated two actin mutants (K853A and I704A) in a constitutively active version of mDia2 containing formin homology domains 1 and 2 (FH1FH2) and found that they still induced stable MTs and bound to the MT TIP proteins EB1 and APC, which have also been implicated in MT stabilization. A dimerization-impaired mutant of mDia2 (W630A) also generated stable MTs in cells. We examined whether FH1FH2mDia2 had direct activity on MTs in vitro and found that it bound directly to MTs, stabilized MTs against cold- and dilution-induced disassembly, and reduced the rates of growth and shortening during MT assembly and disassembly, respectively. These results indicate that mDia2 has a novel MT stabilization activity that is separate from its actin nucleation activity.

Regulated binding of adenomatous polyposis coli protein to actin.

Adenomatous polyposis coli (APC) protein is a large tumor suppressor that is truncated in most colorectal cancers. The carboxyl-terminal third of APC protein mediates direct interactions with microtubules and the microtubule plus-end tracking protein EB1. In addition, APC has been localized to actin-rich regions of cells, but the mechanism and functional significance of this localization have remained unclear. Here we show that purified carboxyl-terminal basic domain of human APC protein (APC-basic) bound directly to and bundled actin filaments and associated with actin stress fibers in microinjected cells. Actin filaments and microtubules competed for binding to APC-basic, but APC-basic also could cross-link actin filaments and microtubules at specific concentrations, suggesting a possible role in cytoskeletal cross-talk. APC interactions with actin in vitro were inhibited by its ligand EB1, and co-microinjection of EB1 prevented APC association with stress fibers. Point mutations in EB1 that disrupted APC binding relieved the inhibition in vitro and restored APC localization to stress fibers in vivo, demonstrating that EB1-APC regulation is direct. Because tumor formation and metastasis involve coordinated changes in the actin and microtubule cytoskeletons, this novel function for APC and its regulation by EB1 may have direct implications for understanding the molecular basis of tumor suppression.

Moesin regulates stable microtubule formation and limits retroviral infection in cultured cells.

In a functional screen of mammalian complementary DNA libraries, we identified moesin as a novel gene whose overexpression blocks infection by murine leukemia viruses and human immunodeficiency virus type 1 in human and rodent lines, before the initiation of reverse transcription. Knockdown of moesin by RNA interference resulted in enhanced infection, suggesting that even the endogenous basal levels of moesin in rat fibroblasts are sufficient to limit virus infection. Moesin acts as a crosslinker between plasma membrane and actin filaments, as well as a signal transducer in responses involving cytoskeletal remodeling. Moesin overexpression was found to downregulate the formation of stable microtubules, whereas knockdown of moesin increased stable microtubule formation. A virus-resistant mutant cell line also displayed decreased stable microtubule levels, and virus-sensitive revertants recovered from the mutant line showed restoration of the stable microtubules, suggesting that these cytoskeletal networks play an important role in early post-entry events in the retroviral lifecycle. Together, these results suggest that moesin negatively regulates stable microtubule networks and is a natural determinant of cellular sensitivity to retroviral infection.

Src SH2 arginine 175 is required for cell motility: specific focal adhesion kinase targeting and focal adhesion assembly function.

Src kinase is a crucial mediator of adhesion-related signaling and motility. Src binds to focal adhesion kinase (FAK) through its SH2 domain and subsequently activates it for phosphorylation of downstream substrates. In addition to this binding function, data suggested that the SH2 domain might also perform an important role in targeting Src to focal adhesions (FAs) to enable further substrate phosphorylations. To examine this, we engineered an R175L mutation in cSrc to prevent the interaction with FAK pY397. This constitutively open Src kinase mediated up-regulated substrate phosphorylation in SYF cells but was unable to promote malignant transformation. Significantly, SrcR175L cells also had a profound motility defect and an impaired FA generation capacity. Importantly, we were able to recapitulate wild-type motile behavior and FA formation by directing the kinase to FAs, clearly implicating the SH2 domain in recruitment to FAK and indicating that this targeting capacity, and not simply Src-FAK scaffolding, was critical for normal Src function.

The sulindac derivatives OSI-461, OSIP486823, and OSIP487703 arrest colon cancer cells in mitosis by causing microtubule depolymerization.

Exisulind (sulindac sulfone) and three highly potent derivatives, OSI-461 (CP461), OSIP486823 (CP248), and OSIP487703, inhibit growth and induce apoptosis in SW480 human colon cancer cells, with IC(50)s of 200, 2, 0.1, and 0.003 micromol/L, respectively. The latter three compounds, but not exisulind, induce marked M-phase cell cycle arrest in these cells. This effect seems to be independent of the known ability of these compounds to cause activation of protein kinase G. When tested at twice their IC(50) concentration for growth inhibition, OSI-461, OSIP486823, and OSIP487703 cause depolymerization of microtubules in interphase cells, inhibit spindle formation in mitotic cells, and induce multinucleated cells. In vitro tubulin polymerization assays indicate that all three compounds interact with tubulin directly to cause microtubule depolymerization and/or inhibit de novo tubulin polymerization. These results suggest that the dual effects of OSI-461, OSIP486823, and OSIP487703 on impairment of microtubule functions and protein kinase G activation may explain the potent antiproliferative and apoptotic effects of these compounds in cancer cells.

Regulation of microtubules by Rho GTPases in migrating cells.

Microtubules (MTs) contribute to cell polarization and migration, but the molecular mechanism involved are unknown. We have explored signalling pathways that generate specific changes in MTs arrays in wounded monolayers of fibroblasts. In earlier work, we found that Rho GTPase and its effector mDia, stimulate selective MT stabilization in the lamella, whereas Cdc42 and the MT motor protein dynein regulate MT organizing centre (MTOC) reorientation towards the leading edge. We have now found that the MT tip proteins EB1 and adenomatous polyposis coli protein (APC) function with mDia to stabilize MTs and interact directly with mDia. EB1, APC and mDia localize to the ends of stabilized MTs suggesting that they may contribute to capping of these MTs. Models of MTOC reorientation suggest that the MTOC moves in front of the nucleus by dynein pulling on MTs. In contrast, we find by directly imaging MTOC reorientation that the nucleus moves rearward while the MTOC remains stationary. Rearward nuclear movement is coupled to retrograde actin-myosin flow and is regulated by Cdc42 and its effector myotonic dystrophy kinase-related Cdc42-binding kinase. Dynein is not involved in nuclear movement, but is essential to maintain the MTOC at the cell centroid. These results show that there are two Cdc42 pathways that regulate MTOC reorientation.

Effects of a series of organosulfur compounds on mitotic arrest and induction of apoptosis in colon cancer cells.

We previously reported that the garlic-derived compound S-allylmercaptocysteine (SAMC) causes growth inhibition, mitotic arrest, and induction of apoptosis in SW480 human colon cancer cells by inducing microtubule depolymerization and c-Jun NH(2) terminus kinase-1 activation. In the present study, we compared the aforementioned effects of SAMC to those of a series of garlic-derived and other organosulfur compounds. Among the 10 compounds tested, only SAMC, diallyl disulfide (DADS), and S-trityl-L-cysteine (trityl-cys) cause significant inhibition of cell growth with IC(50) values of 150, 56, and 0.9 micromol/L, respectively. These three compounds also induce G(2)-M cell cycle arrest and apoptosis. Further studies reveal that, like SAMC, the garlic-derived compound DADS exerts antiproliferative effects by binding directly to tubulin and disrupting the microtubule assembly, thus arresting cells in mitosis and triggering mitochondria-mediated signaling pathways that lead to apoptosis. However, the synthetic compound trityl-cys exerts its effect on M-phase arrest and growth inhibition by mechanisms that involve spindle impairment but do not involve disruption of microtubule structure or dynamics. Furthermore, trityl-cys does not induce marked loss of mitochondrial membrane potential or release of cytochrome c, but it does induce caspase-3 activation and poly(ADP-ribose) polymerase cleavage. Structure-function analysis suggests that both the allyl and the disulfide moieties are important features for the antiproliferative effects of SAMC and DADS. These findings may be useful in the identification, synthesis, and development of organosulfur compounds that have anticancer activity.