Olga N. Zhapparova

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

This study shows that the signal transduction pathway responsible for the initiation of minus-end–directed movement of membrane-bounded pigment granules in melanophores involves sequential activation of protein phosphatase 2A and casein kinase 1 and that this activation correlates with increased phosphorylation of the dynein intermediate chain.

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 motors 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.

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 (t1/2 ∼ 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.

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.

The centrosome keeps nucleating microtubules but looses the ability to anchor them after the inhibition of dynein-dynactin complex.

We inhibited dynein in cells either by the expression of coiled coil-1 (CC1) fragment of dynactin p150Glued subunit or by the microinjection of CC1 protein synthesized in Escherichia coli. CC1 impeded the aggregation of pigment granules in fish melanophores and caused the dispersion of Golgi in Vero and HeLa cells. These data demonstrated the inhibiting effect of CC1 on dynein. In cultured cells, CC1 expression caused the disruption of microtubule array, while the nucleation of new microtubules remained unaltered. This was proved both with in vivo microtubule recovery after nocodazole treatment and with in vitro microtubule polymerization on centrosomes, when the number of nucleated microtubules marginally reduced after the incubation with CC1. Moreover, the inhibiting anti-dynein 74.1 antibodies caused the same effect. Thus we have shown that though dynein is not important for microtubule nucleation, it is essential for the radial organization of microtubules presumably being involved in microtubule anchoring on the centrosome.

Ste20-like Protein Kinase SLK (LOSK) Regulates Microtubule Organization by Targeting Dynactin to the Centrosome

The protein kinase SLK (LOSK) phosphorylates the 1A isoform of the p150Glued subunit of dynactin and targets it to the centrosome, where it maintains microtubule radial organization. In addition, dynactin phosphorylation is involved in Golgi reorientation in polarized cells.

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.

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 IVIGs 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.