Cytoskeleton/endoplasmic reticulum collapse induced by prostaglandin J2 parallels centrosomal deposition of ubiquitinated protein aggregates

Many neurodegenerative disorders, such as Parkinson disease, exhibit inclusion bodies containing ubiquitinated proteins. The mechanisms implicated in this aberrant protein deposition remain elusive. In these disorders signs of inflammation are also apparent in the affected central nervous system areas. We show that prostaglandin J2 (PGJ2), an endogenous product of inflammation, disrupts the cytoskeleton in neuronal cells. Furthermore, PGJ2 perturbed microtubule polymerization in vitro and decreased the number of free sulfhydryl groups on tubulin cysteines. A direct effect of PGJ2 on actin was not apparent, although actin filaments were altered in cells treated with PGJ2. This cyclopentenone prostaglandin triggered endoplasmic reticulum (ER) collapse and the redistribution of ER proteins, such as calnexin and catechol-O-methyltransferase, into a large centrosomal aggregate containing ubiquitinated proteins and alpha-synuclein. The PGJ2-dependent cytoskeletal rearrangement paralleled the development of the large centrosomal aggregate. Both of these events were replicated by treating cells with colchicine, which disrupts the microtubule/ER network, but not with brefeldin A, which impairs ER/Golgi transport. PGJ2 also perturbed 26 S proteasome assembly and activity, which preceded the accumulation of ubiquitinated proteins as detergent/salt-insoluble aggregates. Our data support a mechanism by which, upon PGJ2 treatment, cytoskeleton/ER collapse coincides with the relocation of ER proteins, other potentially neighboring proteins, and ubiquitinated proteins into centrosomal aggregates. Development of these large perinuclear aggregates is associated with disruption of the microtubule/ER network. This aberrant protein deposition, triggered by a product of inflammation, may be common to other compounds that disrupt microtubules and induce protein aggregation, such as MPP+ and rotenone, found to be associated with neurodegeneration.     

Lis1 is essential for cortical microtubule organization and desmosome stability in the epidermis

Desmosomes are cell-cell adhesion structures that integrate cytoskeletal networks. In addition to binding intermediate filaments, the desmosomal protein desmoplakin (DP) regulates microtubule reorganization in the epidermis. In this paper, we identify a specific subset of centrosomal proteins that are recruited to the cell cortex by DP upon epidermal differentiation. These include Lis1 and Ndel1, which are centrosomal proteins that regulate microtubule organization and anchoring in other cell types. This recruitment was mediated by a region of DP specific to a single isoform, DPI. Furthermore, we demonstrate that the epidermal-specific loss of Lis1 results in dramatic defects in microtubule reorganization. Lis1 ablation also causes desmosomal defects, characterized by decreased levels of desmosomal components, decreased attachment of keratin filaments, and increased turnover of desmosomal proteins at the cell cortex. This contributes to loss of epidermal barrier activity, resulting in completely penetrant perinatal lethality. This work reveals essential desmosome-associated components that control cortical microtubule organization and unexpected roles for centrosomal proteins in epidermal function.     

Cep70 promotes microtubule assembly in vitro by increasing microtubule elongation

Microtubules are dynamic cytoskeletal polymers present in all eukaryotic cells. In animal cells, they are organized by the centrosome, the major microtubule-organizing center. Many centrosomal proteins act coordinately to modulate microtubule assembly and organization. Our previous work has shown that Cep70, a novel centrosomal protein regulates microtubule assembly and organization in mammalian cells. However, the molecular details remain to be investigated. In this study, we investigated the molecular mechanism of how Cep70 regulates microtubule assembly using purified proteins. Our data showed that Cep70 increased the microtubule length without affecting the microtubule number in the purified system. These results demonstrate that Cep70 could directly regulate microtubule assembly by promoting microtubule elongation instead of microtubule nucleation.     

Centrosomes and cancer: lessons from a TACC

The recent discovery that many cancer cells have centrosomal abnormalities suggests a link between centrosomes and cancer. Members of the transforming acidic coiled-coil (TACC) family of proteins have been implicated in cancer and are concentrated at centrosomes, where they regulate microtubule stability. I discuss a model of how the TACC proteins might contribute to cancer. This model predicts that defects in TACC function can make important contributions to the development of cancer but are unlikely to be the primary cause of cancer. The model might also apply to several other centrosomal proteins that have been linked to cancer.     

AKAP350 modulates microtubule dynamics

AKAP350 is a multiply spliced type II protein kinase A-anchoring protein that localizes to the centrosomes in most cells and the Golgi apparatus in epithelial cells. Multiple studies suggest that AKAP350 is involved in microtubule nucleation at the centrosome. Our previous studies demonstrated that AKAP350 was necessary for the maintenance of Golgi apparatus integrity. These data suggested that AKAP350 might be necessary for normal cytoskeletal interactions with the Golgi. To examine the relationship of AKAP350 with the microtubule cytoskeleton, we analyzed the effect of the depletion of AKAP350 on microtubule regrowth after nocodazole treatment in HeLa cells. The decrease in AKAP350 expression with short interfering RNA induced a delay in microtubule elongation with no effect on microtubule aster formation. In contrast, overexpression of the centrosomal targeting domain of AKAP350 elicited alterations in aster formation, but did not affect microtubule elongation. RNA interference for AKAP350 also induced an increase in cdc42 activity during microtubule regrowth. Our data suggest that AKAP350 has a role in the remodeling of the microtubule cytoskeleton.     

A human centrosomal protein is immunologically related to basal body- associated proteins from lower eucaryotes and is involved in the nucleation of microtubules

Isolation of centrosomes from human cells has revealed a proteic pattern which is both complex and specific. As the most prominent structural element of centrosomes in animal cells, the centriole which is present as two copies, is a highly conserved structure, we have attempted to identify centrosomal proteins on the basis of immunocross-reaction with proteins identified in basal bodies from lower eucaryotes. We report that two antibodies, one raised against the Ca(+)-binding protein centrin (Salisbury, J. L., A. T. Baron, B. Surek, and M. Melkonian. 1984. J. Cell Biol. 99:962-970) and the other directed against a 230-kD protein isolated from the infraciliary cytoskeletal lattice of the protozoan Polyplastron m., decorate the centrosome of human cultured cells, and identify one of the major centrosomal components revealed as a doublet of 62/64 kD. Moreover the nucleation reaction of microtubules, which can be efficiently produced on isolated centrosomes, is blocked by the antibodies, a result which strongly implicates the 62/64-kD protein in this centrosomal activity. We also show that the 62/64-kD protein remains insoluble in conditions (0.5 M KI or 8 M urea) which are capable of extracting most of the centrosomal proteins. Immunocytochemical localization by EM of isolated centrosomes revealed the association of this 62/64-kD doublet with the intercentriolar link and the pericentriolar lattice. Our results suggest that conservation of structure in the centrosome from divergent organisms could be matched by conservation of proteins and activity, evidence for the maintenance of a specific function, which could involve Ca2+, associated with the microtubule organizing centers.     

p21(Cip1) regulates cell-substrate adhesion and interphase microtubule dynamics in untransformed human mammary epithelial cells

Despite its frequent inactivation in human breast cancers, the role of p21(Cip1) (p21) in morphological plasticity of normal mammary epithelial cells is still poorly understood. To address this question, we have investigated the consequences of p21 silencing in two-dimensional (2D) morphogenesis of untransformed human mammary epithelial cells. Here we show that p21 inactivation causes a reduction of 2D cell spreading and suppresses focal adhesion. In order to investigate the cytoskeletal modifications associated with this altered morphology, we have analyzed the microtubule dynamics in interphase p21-depleted cells. Our results demonstrate that interphase microtubule dynamic instability is strongly increased by p21 silencing. This alteration correlates with severe microtubule hypoacetylation. Next, we show that these microtubule defects in p21-depleted cells can be reversed by the use of the small molecule tubacin, a specific inhibitor of the α-tubulin deacetylase HDAC6. Tubacin-induced microtubule dynamics decrease also correlates with a partial recovery of cell spreading and focal adhesion in those cells. Collectively, these data indicate that p21 regulates the morphological plasticity of normal mammary epithelial cells by modulating dynamics of key cytoskeletal components.     

The role of calpain in the proteolytic cleavage of E-cadherin in prostate and mammary epithelial cells

The E-cadherin protein mediates Ca(2+)-dependent interepithelial adhesion. Association of E-cadherin with the catenin family of proteins is critical for the maintenance of a functional adhesive complex. We have identified a novel truncated E-cadherin species of 100-kDa (E-cad(100)) in prostate and mammary epithelial cells. E-cad(100) was generated by treatment of cells with ionomycin or TPA. Cell-permeable calpain inhibitors prevented E-cad(100) induction by ionomycin. Immunoblotting for spectrin and mu-calpain confirmed calpain activation in response to ionomycin treatment. Both the mu- and m-isoforms of calpain efficiently generated E-cad(100) in vitro. The E-cad(100) fragment was unable to bind to beta-catenin, gamma-catenin, and p120, suggesting that this cleavage event would disrupt the E-cadherin adhesion complex. Mutational analysis localized the calpain cleavage site to the cytosolic domain upstream of the beta- and gamma-catenin binding motifs of E-cadherin. Because E-cadherin is inactivated in many adenocarcinomas we hypothesized that calpain may play a role in prostate tumorigenesis. A prostate cDNA microarray data base was analyzed for calpain expression in which it was found that m-calpain was up-regulated in localized prostate cancer, and to an even higher degree in metastatic prostate cancer compared with normal prostate tissue. Furthermore, we examined the cleavage of E-cadherin in prostate cancer specimens and found that E-cad(100) accumulated in both localized and metastatic prostate tumors, supporting the cDNA microarray data. These findings demonstrate a novel mechanism by which E-cadherin is functionally inactivated through calpain-mediated proteolysis and suggests that E-cadherin is targeted by calpain during the tumorigenic progression of prostate cancer.     

Arabidopsis TONNEAU1 proteins are essential for preprophase band formation and interact with centrin

Plant cells have specific microtubule structures involved in cell division and elongation. The tonneau1 (ton1) mutant of Arabidopsis thaliana displays drastic defects in morphogenesis, positioning of division planes, and cellular organization. These are primarily caused by dysfunction of the cortical cytoskeleton and absence of the preprophase band of microtubules. Characterization of the ton1 insertional mutant reveals complex chromosomal rearrangements leading to simultaneous disruption of two highly similar genes in tandem, TON1a and TON1b. TON1 proteins are conserved in land plants and share sequence motifs with human centrosomal proteins. The TON1 protein associates with soluble and microsomal fractions of Arabidopsis cells, and a green fluorescent protein–TON1 fusion labels cortical cytoskeletal structures, including the preprophase band and the interphase cortical array. A yeast two-hybrid screen identified Arabidopsis centrin as a potential TON1 partner. This interaction was confirmed both in vitro and in plant cells. The similarity of TON1 with centrosomal proteins and its interaction with centrin, another key component of microtubule organizing centers, suggests that functions involved in the organization of microtubule arrays by the centrosome were conserved across the evolutionary divergence between plants and animals.     

AKAP220 protein organizes signaling elements that impact cell migration

Cell movement requires the coordinated reception, integration, and processing of intracellular signals. We have discovered that the protein kinase A anchoring protein AKAP220 interacts with the cytoskeletal scaffolding protein IQGAP1 to influence cell motility. AKAP220/IQGAP1 networks receive and integrate calcium and cAMP second messenger signals and position signaling enzymes near their intended substrates at leading edges of migrating cells. IQGAP1 supports calcium/calmodulin-dependent association of factors that modulate microtubule dynamics. AKAP220 suppresses GSK-3β and positions this kinase to allow recruitment of the plus-end microtubule tracking protein CLASP2. Gene silencing of AKAP220 alters the rate of microtubule polymerization and the lateral tracking of growing microtubules and retards cell migration in metastatic human cancer cells. This reveals an unappreciated role for this anchored kinase/microtubule effector protein network in the propagation of cell motility.     

Ecto-5'-nucleotidase (eN, CD73) is coexpressed with metastasis promoting antigens in human melanoma cells

Upregulated expression of eN has been found in the highly invasive human melanoma cell lines but neither in melanocytes nor in primary tumor cells. Membrane proteins associated with cell adhesion and metastasis: alpha5-, beta1-, beta3-integrins, and CD44 were elevated gradually in accordance with increasing metastatic potential. alphav-integrin was seen mostly in aggressive melanomas. The expression of eN correlated with a number of metastasis-related markers and thus may have a function in the process. eN activity went parallel with its amount in all cells. Concanavalin A strongly inhibited the enzyme in a noncompetitive way. Clustering of eN protein in overexpressing cells by ConA-treatment increased the enzyme association with the heavy cytoskeletal complexes. A similar shift towards cytoskeletal fractions took also place with other membrane proteins coexpressed with eN. This ConA-induced association may reflect a putative interaction of eN with physiological ligand, that upon interaction, aggregates protein components of lipid rafts and triggers signaling pathway that may be intrinsically involved in cell-stroma adhesion.     

Attachment of A-431 cells on immobilized antibodies to the EGF receptor promotes cell spreading and reorganization of the microfilament system

EGF-like sequences, inherent in a number of extracellular matrix proteins, participate in cell adhesion. It is possible that interactions of these sequences with EGF receptors (EGFR) affect actin filament organization. It was shown previously [Khrebtukova et al., 1991: Exp. Cell Res. 194:48-55] that antibodies specific to EGFR induce capping of these receptors and redistribution of cytoskeletal proteins in A-431 cells. Here we report that A-431 cells attach and spread on solid substrata coated with antibodies to EGFR, even in the absence of serum. Thus, EGFR can act as an adhesion protein and promote microfilament reorganization. Binding of the cells to the EGFR-antibody resulted in the formation of a unique cell shape characterized by numerous, actin-based filopodia radiating from the cell body, but without membrane ruffles. There was also a conspicuous circular belt of actin-containing fibers inside the cell margin, and many irregular actin aggregates in the perinuclear area. The morphologies and actin distributions in A-431 cells spread on fibronectin or laminin 2/4 were very different. On fibronectin, cells had polygonal shapes with numerous stress-fibers and thick actin-containing fibers along the cell edges. On laminin-covered substrata, the cells became fusiform and acquired broad leading lamellae with ruffles. In these cells, there were also a few bundles of filaments running the whole length of the cell body, and shorter bundles extending through the leading lamellae towards the membrane ruffles in the cell edge. These effects and those seen with immobilized EGF suggest that different ligand/receptor complexes induce specific reorganizations of the microfilament system.     

Interaction in vivo and in vitro of the metastasis-inducing S100 protein, S100A4 (p9Ka) with S100A1

The calcium-binding protein S100A4 (p9Ka) has been shown to cause a metastatic phenotype in rodent mammary tumor cells and in transgenic mouse model systems. mRNA for S100A4 (p9Ka) is present at a generally higher level in breast carcinoma than in benign breast tumor specimens, and the presence of immunocytochemically detected S100A4 correlates strongly with a poor prognosis for breast cancer patients. Recombinant S100A4 (p9Ka) has been reported to interact in vitro with cytoskeletal components and to form oligomers, particularly homodimers in vitro. Using the yeast two-hybrid system, a strong interaction between S100A4 (p9Ka) and another S100 protein, S100A1, was detected. Site-directed mutagenesis of conserved amino acid residues involved in the dimerization of S100 proteins abolished the interactions. The interaction between S100A4 and S100A1 was also observed in vitro using affinity column chromatography and gel overlay techniques. Both S100A1 and S100A4 can occur in the same cultured mammary cells, suggesting that in cells containing both proteins, S100A1 might modulate the metastasis-inducing capability of S100A4.     

Cytoskeletal organization and cell motility correlates with metastatic potential and state of differentiation in prostate cancer.

The actin cytoskeleton is the key cellular machinery responsible for cellular movement. Changes in the organization and distribution of actin and actin binding protein are necessary for several cellular processes such as focal adhesion formation, cell motility and cell invasion. Here we examined differences in cytoskeletal protein distribution, cell morphometry and cell motility of metastatic and non-metastatic cells. Correlations were found between metastatic potential phenotypic properties such as cell motility, cell spreading and cytoskeletal organization in prostate cancer. As a cell progresses from a normal state to a malignant state, it loses its ability to function normally and also become poorly differentiated. Differentiation therapy is concerned with the redirection of malignant cells toward a terminal, non-dividing state using non-cytotoxic agents. Two well acknowledged differentiation agents, retinoic acid (RA) and diflouromethylomithine (DFMO) were examined for their ability to alter cellular phenotypes associated with metastatic potential in rat prostate cancer cell lines. The results of these studies indicate that there are sub-cellular differences between non-metastatic and highly metastatic cells relative to cytoskeletal organization. We also show that treatment of highly metastatic cells with either RA or DFMO significantly alters cell morphology, cell morphometry and motility to states similar to non-metastatic cells.     

Ezrin regulates focal adhesion and invadopodia dynamics by altering calpain activity to promote breast cancer cell invasion

Up-regulation of the cytoskeleton linker protein ezrin frequently occurs in aggressive cancer types and is closely linked with metastatic progression. However, the underlying molecular mechanisms detailing how ezrin is involved in the invasive and metastatic phenotype remain unclear. Here we report a novel function of ezrin in regulating focal adhesion (FA) and invadopodia dynamics, two key processes required for efficient invasion to occur. We show that depletion of ezrin expression in invasive breast cancer cells impairs both FA and invadopodia turnover. We also demonstrate that ezrin-depleted cells display reduced calpain-mediated cleavage of the FA and invadopodia-associated proteins talin, focal adhesion kinase (FAK), and cortactin and reduced calpain-1–specific membrane localization, suggesting a requirement for ezrin in maintaining proper localization and activity of calpain-1. Furthermore, we show that ezrin is required for cell directionality, early lung seeding, and distant organ colonization but not primary tumor growth. Collectively our results unveil a novel mechanism by which ezrin regulates breast cancer cell invasion and metastasis.     

Ecto-5′-Nucleotidase (eN,CD73) is Coexpressed with Metastasis Promoting Antigens in Human Melanoma Cells

Upregulated expression of eN has been found in the highly invasive human melanoma cell lines but neither in melanocytes nor in primary tumor cells. Membrane proteins associated with cell adhesion and metastasis: α₅-, β₁-, β₃-integrins, and CD44 were elevated gradually in accordance with increasing metastatic potential. α_v-integrin was seen mostly in aggressive melanomas. The expression of eN correlated with a number of metastasis-related markers and thus may have a function in the process. eN activity went parallel with its amount in all cells. Concanavalin A strongly inhibited the enzyme in a noncompetitive way. Clustering of eN protein in overexpressing cells by ConA-treatment increased the enzyme association with the heavy cytoskeletal complexes. A similar shift towards cytoskeletal fractions took also place with other membrane proteins coexpressed with eN. This ConA-induced association may reflect a putative interaction of eN with physiological ligand, that upon interaction, aggregates protein components of lipid rafts and triggers signaling pathway that may be intrinsically involved in cell-stroma adhesion.     

Asymmetric CLASP-dependent nucleation of noncentrosomal microtubules at the trans-Golgi network

Proper organization of microtubule arrays is essential for intracellular trafficking and cell motility. It is generally assumed that most if not all microtubules in vertebrate somatic cells are formed by the centrosome. Here we demonstrate that a large number of microtubules in untreated human cells originate from the Golgi apparatus in a centrosome-independent manner. Both centrosomal and Golgi-emanating microtubules need gamma-tubulin for nucleation. Additionally, formation of microtubules at the Golgi requires CLASPs, microtubule-binding proteins that selectively coat noncentrosomal microtubule seeds. We show that CLASPs are recruited to the trans-Golgi network (TGN) at the Golgi periphery by the TGN protein GCC185. In sharp contrast to radial centrosomal arrays, microtubules nucleated at the peripheral Golgi compartment are preferentially oriented toward the leading edge in motile cells. We propose that Golgi-emanating microtubules contribute to the asymmetric microtubule networks in polarized cells and support diverse processes including post-Golgi transport to the cell front.     

Human Cep192 is required for mitotic centrosome and spindle assembly

As cells enter mitosis, centrosomes dramatically increase in size and ability to nucleate microtubules. This process, termed centrosome maturation, is driven by the accumulation and activation of gamma-tubulin and other proteins that form the pericentriolar material on centrosomes during G2/prophase. Here, we show that the human centrosomal protein, Cep192 (centrosomal protein of 192 kDa), is an essential component of the maturation machinery. Specifically, we have found that siRNA depletion of Cep192 results in a complete loss of functional centrosomes in mitotic but not interphase cells. In mitotic cells lacking Cep192, microtubules become organized around chromosomes but rarely acquire stable bipolar configurations. These cells contain normal numbers of centrioles but cannot assemble gamma-tubulin, pericentrin, or other pericentriolar proteins into an organized PCM. Alternatively, overexpression of Cep192 results in the formation of multiple, extracentriolar foci of gamma-tubulin and pericentrin. Together, our findings support the hypothesis that Cep192 stimulates the formation of the scaffolding upon which gamma-tubulin ring complexes and other proteins involved in microtubule nucleation and spindle assembly become functional during mitosis.     

Microtubule release and capture in epithelial cells.

Many differentiated cells including polarised epithelial cells display a non-radial, apico-basal microtubule array. In some cells the centrosome disassembles and new nucleating sites are created at more appropriate locations. In others the centrosome remains, but relatively few microtubules radiate from it's immediate environs. Instead, the majority of the microtubule minus-ends are associated with apical cell surface sites. Centrosomal microtubule release and capture is evidently a mechanism exploited by some polarised epithelial cells as a means of producing non-centrosomal, apico-basal microtubule arrays. This involves microtubule nucleation at the centrosome, release and subsequent translocation and capture at the apical sites. Two functionally distinct centrosomal complexes dedicated to the control of microtubule nucleation and anchorage have been suggested to be essential and universal features of all centrosomes. The centrosomal proteins ninein and R2 are potential microtubule anchoring proteins and their discovery has exciting implications for centrosomal organisation and microtubule positioning in cells.