N.A. Shanina

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

BackgroundYB-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.ResultsWe 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.ConclusionThese 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.

A new ATPase in cytoplasmic microtubule preparations

Microtubules, cylindrical structures of about 250 A in diameter, are present in the cytoplasm of most eukaryotic cells. Although their functions are still obscure, there are grounds for believing that they play a significant role in intracellular motion. This follows, in particular, from the fact that the breakdown of microtubules (e.g., by colchicine) results in cessation of divergence of chromosomes in mitosis [ 1 ], of axoplasmic transport [2], of saltatory movement of intracellular granules [3] and of some other types of motion. If microtubules directly participate in cytoplasmic transport, it is natural to assume that they contain ATPase whose role is mechanoehemical coupling. Such ATPase is well known in the case of cilia and flagella: it is dynein, a protein with a mol. wt > 300 000, inherent in microtubules of axoneme outer doublets [4]. The present work demonstrates that microtubule preparation isolated from bovine brain by polymerization-depolymerization procedure contains ATPase. This ATPase is capable of cosedimentation with microtubules. Its activity is not suppressed by membrane ATPase inhibitors, neither is it due to the presence in the preparation of actin or myosin impurities. Possibly, the ATPase revealed is inherent to microtubules in rive and concerned with mechanochemical coupling.

MAP2‐mediated binding of chromaffin granules to microtubules

We have examined the interaction of chromaffin granules from bovine adrenal modulla with microtubules. Chromaffin granules were mixed with microtubules made of phosphocellulose‐purified tubulin, and pelleted through a 1.6 M sucrose cushion at 12000 × g for 10 min. Both components (granules and microtubules) were pelleted when added together but not separately. This result indicates that granules form a heavy complex with the microtubules. Such a complex was visualized by an electron microscopy of the granule/microtubule mixture. Treatment of the granules with trypsin abolished their ability to interact with the microtubules. The binding of the granules to the microtubules: (i) was not sensitive to ATP: and (ii) was completely inhibited by the cleavage of C‐terminal peptides of α‐ and β‐subunits of tubulin with subtilisin. These relationships suggest that the granule binding is mediated by one of the structural microtubule‐associated proteins rather than by microtubule‐dependent translocators. For identification of protein(s) mediating the binding, the granules were solubilized with Triton X‐100, soluble proteins were mixed with the microtubules, and microtubules with bound proteins were pelleted through a glycerol cushion. At least one granule protein interacting with the microtubules was found in the pellet. This protein was identified as MAP2 according to its electrophoretic mobility and reactivity with a MAP2 antibody. Affinity chromatography of solubilized proteins on a column containing taxol‐stabilized microtubules also revealed MAP2 as a protein of chromaffin granules interacting with the 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.

Translation initiation factor eIF3 probably binds with microtubules in mammalian cells

Association of the translation apparatus with the cytoskeleton is essential for its transportation within the cell and probably also for translation regulation. Very little is known about the involvement of particular proteins of this association. A polypeptide homologous with the heavy chain of translation initiation factor eIF3 p170 was found earlier in a microtubule preparation from adrenal cells. Antibody A167 directed against the recombinant fragment of p170 has been generated to study eIF3 interaction with microtubules in mammalian cells. This antibody was shown to recognize a single 170-kDa polypeptide in eIF3 preparations as well as in homogenates of various cell types. A167 allowed detection of the 170-kDa polypeptide in microtubule preparation from bovine brain and confirmation of its presence in microtubule preparations from adrenal cells. As shown by immunofluorescence microscopy using A167, the 170-kDa polypeptide is mainly located in the endoplasm within numerous small and some large granules. Cell treatment with cycloheximide resulted in growth and clustering of the large granules, and partial antigen redistribution along intracellular microtubules. These new experimental data indicate that mammalian translation factor eIF3 may bind with microtubules.

Virions and the Coat Protein of the Potato Virus X Interact with Microtubules and Induce Tubulin Polymerization In Vitro

A study was made of the in vitro interactions of virions and the coat protein (CP) of the potato virus X (PVX) with microtubules (MT). Both virions and CP cosedimented with taxol-stabilized MT. In the presence of PVX CP, tubulin polymerized to produce structures resistant to chilling. Electron microscopy revealed the aberrant character of the resulting tubulin polymers (protofilaments and their sheets), which differed from MT assembled in the presence of cell MAP2. In contrast, PVX virions induced the assembly of morphologically normal MT sensitive to chilling. Virions were shown to compete with MAP2 for MT binding, suggesting an overlap for the MT sites interacting with MAP2 and with PVX virions. It was assumed that PVX virions interact with MT in vivo and that, consequently, cytoskeleton elements participate in intracellular compartmentalization of the PVX genome.

A high molecular weight polypeptide cross-reacting with the antibodies to the dynein heavy chain localizes to the subset of Golgi complex in higher plant cells.

Antibodies were produced against fragments of the microtubule‐binding domain and the motor domain of the dynein heavy chain from Dictyostelium discoideum to probe whole cell extracts of root meristem cells of wheat Triticum aestivum. In plant extracts, these antibodies cross‐reacted with a polypeptide of high molecular weight (>500 kDa). The antibodies bound to protein A‐Sepharose precipitated high molecular weight polypeptide from cell extracts. Immunofluorescence showed that the antibodies identified various aggregates inside cells, localized at the perinuclear area during interphase to early prophase, at the spindle periphery and polar area during mitosis, and in the interzonal region during phragmoplast development. Some aggregates were also co‐labeled by markers for the Golgi apparatus. Thus, we found in higher plant cells a high molecular weight antigen cross‐reacting with the antibodies to motor and microtubule‐binding domains of dynein heavy chains. This antigen is associated with aggregates distributed in the cytoplasm in cell cycle‐dependent manner. A subset of these aggregates belongs to the Golgi complex.

Potato virus X coat protein interacts with microtubules in vitro

The cytoskeleton plays an important role in a wide variety of biological activities including intracellular trafficking of animal viruses (Sodeik, 2000). Plant virus transport systems recruit and modify normal plant intraand intercellular pathways for movement of viral proteins and nucleic acids (Jackson, 2000). Virus movement is an active process that requires specific virus-encoded proteins (movement proteins MPs). In some cases additional virus proteins can be involved in virus movement, namely the coat proteins (CPs). Colocalization of tobacco mosaic virus (TMV) 30-kDa MP and TMV RNA with microtubules (MTs) and microfilaments in infected cells and genetic studies led to the suggestion that the interactions between viral MP (viral movement complexes) and MTs are involved in intercellular and cell-to-cell movement of virus genome in infected plants (Boyko et al., 2000). In this study we investigated the interactions between the viral proteins and MTs for potexvirus potato virus X (PVX). This virus requires four proteins for its movement: CP and three MPs encoded by a module of partially overlapping genes. To test the possible interactions between PVX CP and MTs in vitro we used two experimental approaches: (1) cosedimentation CP with paclitaxel-stabilized MTs from bovine brain through glycerol cushion and (2) induction of MT assembly in vitro. It was shown that recombinant PVX CP and native CP purified from PVX virions bind MTs and were detected in the MT pellet (Fig. 1A, line 1). When the MT suspension was preincubated with CP and sedimented through the glycerol cushion saturated with MAP2 the pellet contained significant amounts of MAP2, whereas the amount of CP decreased remarkably (Fig. 1A, line 3). Usually MT-associated proteins promote and enhance MT assembly in vitro. In our experiments purified tubulin did not polymerize if warmed in the spectrophotometer cuvette. The addition of glycerol caused a rapid increasing of turbidity at 330 nm and generation of the typical MT-polymerization curve. This polymerization was cold-reversible. When we added PVX CP in tubulin solution, a rapid increasing of turbidity was detected as well. But there was no cold-dependent reverse in this case (Fig. 1B). Examination of the morphology of PVX CP-induced tubulin polymers using electron microscopy showed that the CP induced the formation of abnormal MTs (open sheets), typical for rapid MT polymerization (Fig. 1C).

A major 170 kDa protein associated with bovine adrenal medulla microtubules: a member of the centrosomin family?

Microtubules isolated from bovine adrenal medulla cells contain a major 170 kDa protein (p170). p170 is heat‐labile and is associated with microtubules in an ATP‐insensitive manner. This protein was purified to near homogeneity using FPLC. A preparation containing purified p170 caused bundling of microtubules. By microsequencing of p170, two polypeptides were identified which appeared to be identical to a recently sequenced p167 centrosomin‐related protein. Polyclonal affinity‐purified anti‐p170 antibody was found to immunostain microtubules and to recognize the 170 kDa polypeptide in culture cells. We suggest that p170 is a new member of a centrosomin family and is a new structural protein associated with microtubules in some cell types.

Purification and some optical characteristics of flagellar ATPase dynein I from sea urchin sperm

According to [ 11, the term ‘dynein’ denotes a class of proteins associated with a system of microtubules of flagella and cilia possessing ATPase activity and high molecular weight of their polypeptide subunits (3-6 X 105). Flagella of sea urchin sperm contain at least two forms of ATPase (dynein I and dynein II) varying in their electrophoretic and immunochemical characteristics [2]. Localization and function of the minor ATPase (dynein II) has not been clarified yet. Dynein I is a better studied protein. It was shown that dynein I is located in the external arms of outer doublet microtubules [3]. It was reported [l] enzymatic and structural evidence in favour of an active role of dynein I in the mechanochemical conjugation at flagellar motility analogous to the role of myosin in muscular contraction. Here we describe the modified method of purification of dynein I from flagella of sea urchin sperm, which allowed us to obtain a homogeneous preparation of purity >90%, we give optical characteristics (absorption spectra at 240-360 run and circular dichroism spectra at 186-3 10 nm) of the purified preparation and estimate the secondary structure of dynein I from circular dichroism spectra.