Divergent Contribution of the Golgi Apparatus to Microtubule Organization in Related Cell Lines.

Membrane trafficking in interphase animal cells is accomplished mostly along the microtubules. Microtubules are often organized radially by the microtubule-organizing center to coordinate intracellular transport. Along with the centrosome, the Golgi often serves as a microtubule-organizing center, capable of nucleating and retaining microtubules. Recent studies revealed the role of a special subset of Golgi-derived microtubules, which facilitates vesicular traffic from this central transport hub of the cell. However, proteins essential for microtubule organization onto the Golgi might be differentially expressed in different cell lines, while many potential participants remain undiscovered. In the current work, we analyzed the involvement of the Golgi complex in microtubule organization in related cell lines. We studied two cell lines, both originating from green monkey renal epithelium, and found that they relied either on the centrosome or on the Golgi as a main microtubule-organizing center. We demonstrated that the difference in their Golgi microtubule-organizing activity was not associated with the well-studied proteins, such as CAMSAP3, CLASP2, GCC185, and GMAP210, but revealed several potential candidates involved in this process.

Centering and Shifting of Centrosomes in Cells.

Centrosomes have a nonrandom localization in the cells: either they occupy the centroid of the zone free of the actomyosin cortex or they are shifted to the edge of the cell, where their presence is justified from a functional point of view, for example, to organize additional microtubules or primary cilia. This review discusses centrosome placement options in cultured and in situ cells. It has been proven that the central arrangement of centrosomes is due mainly to the pulling microtubules forces developed by dynein located on the cell cortex and intracellular vesicles. The pushing forces from dynamic microtubules and actomyosin also contribute, although the molecular mechanisms of their action have not yet been elucidated. Centrosomal displacement is caused by external cues, depending on signaling, and is drawn through the redistribution of dynein, the asymmetrization of microtubules through the capture of their plus ends, and the redistribution of actomyosin, which, in turn, is associated with basal-apical cell polarization.

Centrosome-derived microtubule radial array, PCM-1 protein, and primary cilia formation.

In animal cells, the centrosome nucleates and anchors microtubules (MT), forming their radial array. During interphase centrosome-derived MT, aster can either team up with other MT network or function in an autonomous manner. What is the function of the centrosome-derived MT aster? We suggested that it might play an important role in the formation of the primary cilium, the organelle obligatorily associated with the centrosome. PCM-1 (PeriCentriolar Matrix 1) protein, which participates in the organization of the primary cilium, is a part of pericentiolar satellites. They are transported to the centrosome along MTs by the motor protein dynein in a complex with its cofactor dynactin. Previously, we showed that SLK/LOSK phosphorylated the p150Glued subunit of dynactin, thus promoting its centrosomal targeting followed by its participation in the retention of microtubules. Here, we found that under the repression of SLK/LOSK activity, the PCM-1 protein lost its pericentrosomal localization and was being dispersed throughout the cytoplasm. Despite that the alanine and glutamine mutants of p150Glued had opposite effects on PCM-1 localization, they associated with PCM-1 to the same extent. The occurrence of primary cilia also significantly decreased when SLK/LOSK was repressed. These defects also correlated with a disturbance of the long-range transport in cells, whereas dynein-depending motility was intact. Treatment with the GSK-3ß kinase inhibitor also resulted in the loss of the centrosome-derived MT aster, dispersion of PCM-1 over the cytoplasm, and reduction of primary cilia occurrence. Thus, kinases involved in the centrosome-derived MT aster regulation can indirectly control the formation of primary cilia in cells.

On the interaction of ribosomal protein RPL22e with microtubules.

Microtubule (MT) protein preparations often contain components of the translation machinery, including ribosome proteins. To understand the biological meaning of it we studied the interaction of ribosomal protein RPL22e with the MT. We found that bacteria expressed purified RPL22e-GFP-6His did co-sediment with brain tubulin MTs with 1.3 µM dissociation coefficient. Such a KD is comparable to some specific MT-associated proteins. Distinct in vitro interaction of RPL22e-GFP with MTs was also observed by TIRF microscopy. In real-time assay, RPL22e-GFP molecules stayed bound to MTs for several seconds, and 15% of them demonstrated random-walk along MTs with diffusion coefficient 0.03 µ2 /s. Deletion of basic areas of RPL22e did not have an impact on KD , and deletion of acidic tail slightly increased association with MTs. Interestingly, the deletion of acidic tail increased diffusion coefficient as well. The interaction of RPL22e with MTs is hardly noticeable in vivo in cultured cells, probably since a significant part of the protein is incorporated into the ribosomes. The mobility of ribosomal protein on the MTs probably prevents its interfering with MT-dependent transport and could ameliorate its transport to the nucleus.

MAST-like protein kinase IREH1 from Arabidopsis thaliana co-localizes with the centrosome when expressed in animal cells.

MAIN CONCLUSION: The similarity of IREH1 (Incomplete Root Hair Elongation 1) and animal MAST kinases was confirmed; IREH1cDNA was cloned while expressing in cultured animal cells co-localized with the centrosome. In mammals and fruit flies, microtubule-associated serine/threonine-protein kinases (MAST) are strongly involved in the regulation of the microtubule system. Higher plants also possess protein kinases homologous to MASTs, but their function and interaction with the cytoskeleton remain unclear. Here, we confirmed the sequence and structural similarity of MAST-related putative protein kinase IREH1 (At3g17850) and known animal MAST kinases. We report the first cloning of full-length cDNA of the IREH1 from Arabidopsis thaliana. Recombinant GFP-IREH1 protein was expressed in different cultured animal cells. It revealed co-localization with the centrosome without influencing cell morphology and microtubule arrangement. Structural N-terminal region of the IREH1 molecule co-localized with centrosome as well.

SLK/LOSK kinase regulates cell motility independently of microtubule organization and Golgi polarization.

Cell motility is an essential complex process that requires actin and microtubule cytoskeleton reorganization and polarization. Such extensive rearrangement is closely related to cell polarization as a whole. The serine/threonine kinase SLK/LOSK is a potential regulator of cell motility, as it phosphorylates a series of cytoskeleton-bound proteins that collectively participate in the remodeling of migratory cell architecture. In this work, we report that SLK/LOSK is an indispensable regulator of cell locomotion that primarily acts through the small GTPase RhoA and the dynactin subunit p150(Glued). Both RhoA and dynactin affect cytoskeleton organization, polarization, and general cell locomotory activity to various extents. However, it seems that these events are independent of each other. Thus, SLK/LOSK kinase effectively functions as a switch that links all of the processes underlying cell motility to provide robust directional movement.

Ste20-like protein kinase SLK (LOSK) regulates microtubule organization by targeting dynactin to the centrosome.

The microtubule- and centrosome-associated Ste20-like kinase (SLK; long Ste20-like kinase [LOSK]) regulates cytoskeleton organization and cell polarization and spreading. Its inhibition causes microtubule disorganization and release of centrosomal dynactin. The major function of dynactin is minus end-directed transport along microtubules in a complex with dynein motor. In addition, dynactin is required for maintenance of the microtubule radial array in interphase cells, and depletion of its centrosomal pool entails microtubule disorganization. Here we demonstrate that SLK (LOSK) phosphorylates the p150(Glued) subunit of dynactin and thus targets it to the centrosome, where it maintains microtubule radial organization. We show that phosphorylation is required only for centrosomal localization of p150(Glued) and does not affect its microtubule-organizing properties: artificial targeting of nonphosphorylatable p150(Glued) to the centrosome restores microtubule radial array in cells with inhibited SLK (LOSK). The phosphorylation site is located in a microtubule-binding region that is variable for two isoforms (1A and 1B) of p150(Glued) expressed in cultured fibroblast-like cells (isoform 1B lacks 20 amino acids in the basic microtubule-binding domain). The fact that SLK (LOSK) phosphorylates only a minor isoform 1A of p150(Glued) suggests that transport and microtubule-organizing functions of dynactin are distinctly divided between the two isoforms. We also show that dynactin phosphorylation is involved in Golgi reorientation in polarized cells.

Association of nucleus and centrosome: magnet or velcro?

A structural link between cell's nucleus and centrosome was proposed years ago. Such a link was suggested to maintain nucleus-centrosome axis, determine polarity of interphase cells and ensure spindle assembly in mitotic cells. The idea of structural link is supported by the facts that centrosomes are usually located in close proximity to the nuclei and remain attached to the nuclei in mildly homogenated cells. However, juxtaposed location can result rather from the tendency of both organelles to occupy central position in cell than from the existence of a specific structural link. Moreover, the nucleus was shown to be transported towards the centrosome along microtubules by dynein bound to nuclear envelope; inhibition of dynein results in the increase of nucleus-centrosome distance. The interaction of both organelles is disturbed in response to actin depolymerisation, although the exact role of actin filaments in this process remains unknown. The link between the nucleus and the centrosome can support simultaneous migration of nuclei and centrosomes in large cells and in syncytia, but its existence in interphase fibroblast-like and epithelia-like cells was not confirmed yet. Further studies include direct visualisation of a specific link between centrosome and nucleus and elucidation of actin role in its formation.

Suppression of injuries caused by a lytic RNA virus (mengovirus) and their uncoupling from viral reproduction by mutual cell/virus disarmament.

Viruses often elicit cell injury (cytopathic effect [CPE]), a major cause of viral diseases. CPE is usually considered to be a prerequisite for and/or consequence of efficient viral growth. Recently, we proposed that viral CPE may largely be due to host defensive and viral antidefensive activities. This study aimed to check the validity of this proposal by using as a model HeLa cells infected with mengovirus (MV). As we showed previously, infection of these cells with wild-type MV resulted in necrosis, whereas a mutant with incapacitated antidefensive ("security") viral leader (L) protein induced apoptosis. Here, we showed that several major morphological and biochemical signs of CPE (e.g., alterations in cellular and nuclear shape, plasma membrane, cytoskeleton, chromatin, and metabolic activity) in cells infected with L(-) mutants in the presence of an apoptosis inhibitor were strongly suppressed or delayed for long after completion of viral reproduction. These facts demonstrate that the efficient reproduction of a lytic virus may not directly require development of at least some pathological alterations normally accompanying infection. They also imply that L protein is involved in the control of many apparently unrelated functions. The results also suggest that the virus-activated program with competing necrotic and apoptotic branches is host encoded, with the choice between apoptosis and necrosis depending on a variety of intrinsic and extrinsic conditions. Implementation of this defensive suicidal program could be uncoupled from the viral reproduction. The possibility of such uncoupling has significant implications for the pathogenesis and treatment of viral diseases.

Human sphingomyelin synthase 1 gene (SMS1): organization, multiple mRNA splice variants and expression in adult tissues.

We have previously characterized the structure of the human MOB gene (TMEM23), which encodes a hypothetical transmembrane protein (Vladychenskaya et al., 2002, 2004). The primary structure of the peptide that we predicted coincided completely with the amino acid sequence of the later identified sphingomyelin synthase 1 protein (SMS1), which catalyses the transfer of a phosphorylcholine moiety from phosphatidylcholine to ceramide, producing sphingomyelin and diacylglycerol (Huitema et al., 2004; Yamaoka et al., 2004). The gene we found was the SMS1 gene. The combination of in silico and RT-PCR data helped us identify and characterize numerous new transcripts of the human SMS1 gene. We identified mRNA isoforms that vary in the 5'-untranslated region (UTR) and encode the full-length protein, and transcripts resulting from alternative combinations of the exons in the coding region of the gene and the 3'-UTR. Comparison of the discovered transcripts' structures with the sequence of human chromosome 10 showed that the human SMS1 gene comprises at least 24 exons. RT-PCR and real-time PCR data showed that the expression patterns of the alternative SMS1 transcripts are tissue specific. Our results indicate that the regulation of SMS1 expression is complex and occurs at the transcriptional, post-transcriptional and translational levels.

Immunolocalization of Picornavirus RNA in infected cells with antibodies to Tyr-pUp, the covalent linkage unit between VPg and RNA.

The genomic RNA of picornaviruses is attached to a small protein (VPg) via a covalent bond between a tyrosine and a 5'-terminal uridine phosphate. The same structure is present in potyvirus and calicivirus families. VPgs play a key role in initiation of viral replication by acting as primers for RNA synthesis. The model compound [N(Ac),CO(NHMe)]Tyr-(5'P→O)Up-O-(CH(2))(6)NH(2) (mCLU), mimicking this 'covalent linkage unit' (CLU) and containing Tyr-pUp was synthesized in solution following the phosphoramidite scheme and used to raise antibodies for studying picornavirus infection. The antibodies recognized CLU-containing mengovirus RNA and showed minimal cross-reactivity with RNAs lacking CLU. Immunofluorescence staining of cells infected with a human rhinovirus demonstrated co-localization of the signals from anti-mCLU and from anti-VPg antibodies. Efficient synthesis of mCLU and anti-mCLU antibodies might be of great utility for investigating viral replication and identifying yet unknown viral and cellular CLU-containing RNA-protein complexes.

Phosphorylation controls autoinhibition of cytoplasmic linker protein-170.

Cytoplasmic linker protein (CLIP)-170 is a microtubule (MT) plus-end-tracking protein that regulates MT dynamics and links MT plus ends to different intracellular structures. We have shown previously that intramolecular association between the N and C termini results in autoinhibition of CLIP-170, thus altering its binding to MTs and the dynactin subunit p150(Glued) (J. Cell Biol. 2004: 166, 1003-1014). In this study, we demonstrate that conformational changes in CLIP-170 are regulated by phosphorylation that enhances the affinity between the N- and C-terminal domains. By using site-directed mutagenesis and phosphoproteomic analysis, we mapped the phosphorylation sites in the third serine-rich region of CLIP-170. A phosphorylation-deficient mutant of CLIP-170 displays an "open" conformation and a higher binding affinity for growing MT ends and p150(Glued) as compared with nonmutated protein, whereas a phosphomimetic mutant confined to the "folded back" conformation shows decreased MT association and does not interact with p150(Glued). We conclude that phosphorylation regulates CLIP-170 conformational changes resulting in its autoinhibition.

Bioinformatic search of plant microtubule-and cell cycle related serine-threonine protein kinases.

A bioinformatic search was carried for plant homologues of human serine-threonine protein kinases involved in regulation of cell division and microtubule protein phosphorylation (SLK, PAK6, PAK7, MARK1, MAST2, TTBK1, TTBK2, AURKA, PLK1, PLK4 and PASK). A number of SLK, MAST2 and AURKA plant homologues were identified. The closest identified homologue of human AURKA kinase was a protein of unknown function, A7PY12/GSVIVT00026259001 from Vitis vinifera (herein named as "STALK", Serine-Threonine Aurora-Like Kinase). Analysis of STALK's three-dimensional structure confirmed its relationship to the subgroup of AURKA-like protein kinases.

Microtubules govern stress granule mobility and dynamics.

Stress granules (SGs) are ribonucleoprotein (RNP)-containing assemblies that are formed in the cytoplasm in response to stress. Previously, we demonstrated that microtubule depolymerization inhibited SG formation. Here, we show that arsenate-induced SGs move throughout the cytoplasm in a microtubule-dependent manner, and microtubules are required for SG disassembly, but not for SG persistence. Analysis of SG movement revealed that SGs exhibited obstructed diffusion on an average, though sometimes SGs demonstrated rapid displacements. Microtubule depolymerization did not influence preformed SG number and size, but significantly reduced the average velocity of SG movement, the frequency of quick movement events, and the apparent diffusion coefficient of SGs. Actin filament disruption had no effect on the SG motility. In cycloheximide-treated cells SGs dissociated into constituent parts that then dissolved within the cytoplasm. Microtubule depolymerization inhibited cycloheximide-induced SG disassembly. However, microtubule depolymerization did not influence the dynamics of poly(A)-binding protein (PABP) in SGs, according to FRAP results. We suggest that the increase of SG size is facilitated by the transport of smaller SGs along microtubules with subsequent fusion of them. At least some protein components of SGs can exchange with the cytoplasmic pool independently of microtubules.

Dynactin subunit p150Glued isoforms notable for differential interaction with microtubules.

Dynactin is a multiprotein complex that enhances dynein activity. The largest dynactin subunit, p150Glued, interacts with microtubules through its N-terminal region that contains a globular cytoskeleton-associated protein (CAP)-Gly domain and basic microtubule-binding domain of unknown structure. The p150Glued gene has a complicated intron-exon structure, and many splice isoforms of p150Glued protein have been predicted. Here we describe novel natural 150 kDa isoforms: the p150Glued-1A isoform, whose basic domain is composed of 41 amino acids, and p150Glued-1B with a basic domain of 21 aa because of the lack of exons 5-7 in the corresponding messenger RNA (mRNA). According to reverse transcriptase-polymerase chain reaction (RT-PCR) and western blot data, p150Glued-1A is expressed in nerve tissues, in cultured cells and in embryonic tissues, while 1B is expressed ubiquitously. Overexpression of GFP-p150Glued-1A and -1B fusion proteins and immunostaining of cultured cells with 1A-specific antibodies show that the p150Glued-1A isoform is distributed along microtubules, whereas 1B is associated with microtubule plus-ends. The higher affinity of the p150Glued-1A isoform for microtubules is confirmed by a co-pelleting assay. In fibroblast-like cells, the interaction of p150Glued-1A with microtubules is less dependent on EB1/EB3 and CLIP170 proteins, compared with p150Glued-1B. In polarized cells, p150Glued-1A decorates microtubules that face the leading edge of the cell. The pattern of p150Glued-1A and p150Glued-1B interaction with microtubules and their tissue-specific expression patterns suggest that these isoforms might be involved in cell differentiation and proliferation.

YB-1 promotes microtubule assembly in vitro through interaction with tubulin and microtubules.

BACKGROUND: YB-1 is a major regulator of gene expression in eukaryotic cells. In addition to its role in transcription, YB-1 plays a key role in translation and stabilization of mRNAs. RESULTS: We show here that YB-1 interacts with tubulin and microtubules and stimulates microtubule assembly in vitro. High resolution imaging via electron and atomic force microscopy revealed that microtubules assembled in the presence of YB-1 exhibited a normal single wall ultrastructure and indicated that YB-1 most probably coats the outer microtubule wall. Furthermore, we found that YB-1 also promotes the assembly of MAPs-tubulin and subtilisin-treated tubulin. Finally, we demonstrated that tubulin interferes with RNA:YB-1 complexes. CONCLUSION: These results suggest that YB-1 may regulate microtubule assembly in vivo and that its interaction with tubulin may contribute to the control of mRNA translation.

Ste20-related protein kinase LOSK (SLK) controls microtubule radial array in interphase.

Interphase microtubules are organized into a radial array with centrosome in the center. This organization is a subject of cellular regulation that can be driven by protein phosphorylation. Only few protein kinases that regulate microtubule array in interphase cells have been described. Ste20-like protein kinase LOSK (SLK) was identified as a microtubule and centrosome-associated protein. In this study we have shown that the inhibition of LOSK activity by dominant-negative mutant K63R-DeltaT or by LOSK depletion with RNAi leads to unfocused microtubule arrangement. Microtubule disorganization is prominent in Vero, CV-1, and CHO-K1 cells but less distinct in HeLa cells. The effect is a result neither of microtubule stabilization nor of centrosome disruption. In cells with suppressed LOSK activity centrosomes are unable to anchor or to cap microtubules, though they keep nucleating microtubules. These centrosomes are depleted of dynactin. Vero cells overexpressing K63R-DeltaT have normal dynactin "comets" at microtubule ends and unaltered morphology of Golgi complex but are unable to polarize it at the wound edge. We conclude that protein kinase LOSK is required for radial microtubule organization and for the proper localization of Golgi complex in various cell types.

Myosin light chain kinase (210 kDa) is a potential cytoskeleton integrator through its unique N-terminal domain.

Recently discovered 210-kDa myosin light chain kinase (MLCK-210) is identical to 108-130 kDa MLCK, the principal regulator of the myosin II molecular motor, except for the presence of a unique amino terminal extension. Our in vitro experiments and transfected cell studies demonstrate that the N-terminal half of MLCK-210 unique tail domain has novel microfilament and microtubule binding activity. Consistent with this activity, the MLCK-210 domain codistributes with microfilaments and microtubules in cultured cells and with soluble tubulin in nocodazole-treated cells. This domain is capable of aggregating tubulin dimers in vitro, causing bundling and branching of microtubules induced by taxol. The N-terminal actin-binding region of MLCK-210 has lower affinity to actin (K(d) = 7.4 microM) than its central D(F/V)RXXL repeat-based actin-binding site and does not protect stress fibers from disassembly triggered by MLCK inhibition in transfected cells. Obtained results suggest that while being resident on microfilaments, MLCK-210 may interact with other cytoskeletal components through its N-terminal domain. Based on available evidence, we propose a model in which MLCK-210 could organize cell motility by simultaneous control of cytoskeleton architecture and actomyosin activation through the novel protein scaffold function of the unique tail domain and the classical MLCK catalytic function of the kinase domain.

Disruption of microtubules inhibits cytoplasmic ribonucleoprotein stress granule formation.

Stress granules are RNP-containing particles arising in the cytoplasm in response to environmental stress. They are dynamic structures assembling and disassembling in the cytoplasm very rapidly. We have studied whether the cytoskeleton is involved in the formation of stress granules. Stress granules were induced in CV-1 cells by sodium arsenate treatment and visualized by immunofluorescent staining with antibodies either to the p170 subunit of eIF3 or to poly(A)-binding protein. Treatment with sodium arsenate for 30-120 min led to assembling of stress granules in a majority of CV-1 cells. Disruption of MT array with nocodazole treatment abolished arsenate-induced formation of stress granules. A similar effect was induced by the microtubule-depolymerizing drug vinblastine, though the influence of the microtubule-stabilizing drug paclitaxel was opposite. Nocodazole treatment did not prevent arsenate-induced phosphorylation of the eIF-2alpha factor, essential for stress granule formation, suggesting that the presence of intact MT array is required for granule assembly. Unexpectedly, treatment of cells with the actin filament-disrupting drug latrunculin B slightly enhanced stress granule formation. We propose that stress granule formation is microtubule-dependent process and likely is facilitated by the motor protein-driven movement of individual stress granule components (e.g., mRNP) along microtubules.