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.

The effect of Intravenous Immunoglobulin (IVIG) on \textit{ex vivo} activation of human leukocytes.

INTRODUCTION: Intravenous immunoglobulin (IVIG) has been widely used to treat various conditions, including inflammatory and autoimmune diseases. IVIG has been shown to have a direct influence on neutrophils, eosinophils and lymphocytes. However, many aspects IVIG's effect on neutrophils activation still remain unknown. OBJECTIVE: To evaluate the effect of IVIG on PMA-induced activation of neutrophils, with and without priming with TNF-α, in a series of in vitro experiments performed on whole blood. RESULTS: Our data coincided with well-known literature indicating that the effect of phorbol 12-myristate 13-acetate (PMA) on human leukocytes includes activation of neutrophils, monocytes and eosinophils, increase of chemiluminescence (CL) and induction of netosis, resulting in assembly of traps. In presence of IVIG (10 mg/mL), CL was reduced by 25% in response to PMA compared to PMA-induced leukocyte activation without IVIG. Decreasing IVIG concentration to 1 mg/mL and below did not inhibit PMA-induced activation of CL.PMA-induced activation after TNF-α priming resulted in approximately 50% increase of amplitude of CL response to PMA. Moreover, maximum CL was reached by minute 5, which was more rapid than in the absence of TNF-α-priming (in this case maximum CL was reached by minute 15).The IVIG concentrations did not affect morphological changes of leukocytes after sequential addition of TNF-α and PMA. IVIG had no effect on leukocyte content and on PMA-induced CL of primed leukocytes.Addition of IVIG under TNF-α priming significantly increased the number of traps in the smears in response to PMA activation. Of note, such an increase in the number of traps was depended on the IVIG concentration in plasma. CONCLUSION: In conclusion, we suggest that IVIG is able to reduce the degradation of traps under priming with TNF-α. Moreover, IVIG might switch the activation of primed leukocytes to netosis.

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.

Bidirectional transport of organelles: unity and struggle of opposing motors.

Bidirectional transport along microtubules is ensured by opposing motor proteins: cytoplasmic dynein that drives cargo to the minus-ends and various kinesins that generally move to the plus-ends of microtubules. Regulation of motor proteins that are simultaneously bound to the same organelle is required to maintain directional transport and prevent pausing of cargo pulled away by motors of opposite polarity. Debates of the recent decade have been focused on two possible mechanisms of such regulation: (i) coordination, which implies that only one type of motors is active at a given time, and (ii) tug-of-war, which assumes that both motors are active at the same time and that direction of transport depends on the outcome of motor's confrontation. The initial idea of coordination has been challenged by observations of simultaneous activity of plus- and minus-end-directed motors applied to the same cargo. Analysis of the available data indicates that coordination and tug-of-war theories rather complement than contradict each other: cargo interacts with two teams of active motors, the resulting direction and the winner team are determined by coordination complexes, but the activity of the loser team is never completely inhibited and remains at some background level. Such persisting activity might enhance the overall efficiency of transport by increasing processivity or helping to overcome the obstacles on microtubule track.

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.

CK1 activates minus-end-directed transport of membrane organelles along microtubules.

Microtubule (MT)-based organelle transport is driven by MT motor proteins that move cargoes toward MT minus-ends clustered in the cell center (dyneins) or plus-ends extended to the periphery (kinesins). Cells are able to rapidly switch the direction of transport in response to external cues, but the signaling events that control switching remain poorly understood. Here, we examined the signaling mechanism responsible for the rapid activation of dynein-dependent MT minus-end-directed pigment granule movement in Xenopus melanophores (pigment aggregation). We found that, along with the previously identified protein phosphatase 2A (PP2A), pigment aggregation signaling also involved casein kinase 1ε (CK1ε), that both enzymes were bound to pigment granules, and that their activities were increased during pigment aggregation. Furthermore we found that CK1ε functioned downstream of PP2A in the pigment aggregation signaling pathway. Finally, we discovered that stimulation of pigment aggregation increased phosphorylation of dynein intermediate chain (DIC) and that this increase was partially suppressed by CK1ε inhibition. We propose that signal transduction during pigment aggregation involves successive activation of PP2A and CK1ε and CK1ε-dependent phosphorylation of DIC, which stimulates dynein motor activity and increases minus-end-directed runs of pigment granules.

Melanophores for microtubule dynamics and motility assays.

Microtubules (MTs) are cytoskeletal structures essential for cell division, locomotion, intracellular transport, and spatial organization of the cytoplasm. In most interphase cells, MTs are organized into a polarized radial array with minus-ends clustered at the centrosome and plus-ends extended to the cell periphery. This array directs transport of organelles driven by MT-based motor proteins that specifically move either to plus- or to minus-ends. Along with using MTs as tracks for cargo, motor proteins can organize MTs into a radial array in the absence of the centrosome. Transport of organelles and motor-dependent radial organization of MTs require MT dynamics, continuous addition and loss of tubulin subunits at minus- and plus-ends. A unique experimental system for studying the role of MT dynamics in these processes is the melanophore, which provides a useful tool for imaging of both dynamic MTs and moving membrane organelles. Melanophores are filled with pigment granules that are synchronously transported by motor proteins in response to hormonal stimuli. The flat shape of the cell and the radial organization of MTs facilitate imaging of dynamic MT plus-ends and monitoring of their interaction with membrane organelles. Microsurgically produced cytoplasmic fragments of melanophores are used to study the centrosome-independent rearrangement of MTs into a radial array. Here we describe the experimental approaches to study the role of MT dynamics in intracellular transport and centrosome-independent MT organization in melanophores. We focus on the preparation of cell cultures, microsurgery and microinjection, fluorescence labeling, and live imaging of MTs.

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.

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.

Cytoplasmic dynein is involved in the retention of microtubules at the centrosome in interphase cells.

Cytoplasmic dynein is known to be involved in the establishment of radial microtubule (MT) arrays. During mitosis, dynein activity is required for tethering of the MTs at the spindle poles. In interphase cells, dynein inhibitors induce loss of radial MT organization; however, the exact role of dynein in the maintenance of MT arrays is unclear. Here, we examined the effect of dynein inhibitors on MT distribution and the centrosome protein composition in cultured fibroblasts. We found that while these inhibitors induced rapid (t(1/2) approximately 20 min) loss of radial MT organization, the levels of key centrosomal proteins or the rates of MT nucleation did not change significantly in dynein-inhibited cells, suggesting that the loss of dynein activity does not affect the structural integrity of the centrosome or its capacity to nucleate MTs. Live observations of the centrosomal activity showed that dynein inhibition enhanced the detachment of MTs from the centrosome. We conclude that the primary role of dynein in the maintenance of a radial MT array in interphase cells consists of retention of MTs at the centrosome and hypothesize that dynein has a role in the MT retention, separate from the delivery to the centrosome of MT-anchoring proteins.