Mrnp41 (Rae 1p) associates with microtubules in HeLa cells and in neurons

Mrnp41 (hRae1p) is an evolutionarily highly conserved protein, which is a potential component of mRNP particles and plays a role in nuclear mRNA export. The protein is mainly localized at the nuclear pore complex, but is also associated with distinct nuclear domains and with a meshwork of numerous small particles in the cytoplasm (Kraemer and Blobel (1997): Proc. Natl. Acad. Sci. USA 91, 1519-1523). We show that the cytoplasmic pattern of mrnp41 is sensitive to treatment with the microtubule (MT)-depolymerizing drug nocodazole which causes disappearance of mrnp41 from the cell periphery and concentration around the nucleus. By immunofluorescence we demonstrate that mrnp41 colocalizes with MT in HeLa cells and displays an MT-like distribution in cultured neurons. Association of mrnp41 with MT is further demonstrated by copurification with MT from pig brain throughout several steps of polymerization and depolymerization. Separation of MT-associated proteins (MAPs) by phosphocellulose (PC) chromatography showed copurification of mrnp41 with MAPs. These data show an association of mrnp41 with MT and, moreover, demonstrate that an intact MT system is necessary for dispersion of mrnp41-containing particles to the cellular periphery. The essential role of mrnp41 in spindle pole separation and cell cycle progression may also be related to its ability to bind to MTs.     

The mammalian Sec6/8 complex interacts with Ca(2+) signaling complexes and regulates their activity

The localization of various Ca(2+) transport and signaling proteins in secretory cells is highly restricted, resulting in polarized agonist-stimulated Ca(2+) waves. In the present work, we examined the possible roles of the Sec6/8 complex or the exocyst in polarized Ca(2+) signaling in pancreatic acinar cells. Immunolocalization by confocal microscopy showed that the Sec6/8 complex is excluded from tight junctions and secretory granules in these cells. The Sec6/8 complex was found in at least two cellular compartments, part of the complex showed similar, but not identical, localization with the Golgi apparatus and part of the complex associated with Ca(2+) signaling proteins next to the plasma membrane at the apical pole. Accordingly, immunoprecipitation (IP) of Sec8 did not coimmunoprecipitate betaCOP, Golgi 58K protein, or mannosidase II, all Golgi-resident proteins. By contrast, IP of Sec8 coimmunoprecipitates Sec6, type 3 inositol 1,4,5-trisphosphate receptors (IP(3)R3), and the Gbetagamma subunit of G proteins from pancreatic acinar cell extracts. Furthermore, the anti-Sec8 antibodies coimmunoprecipitate actin, Sec6, the plasma membrane Ca(2+) pump, the G protein subunits Galphaq and Gbetagamma, the beta1 isoform of phospholipase C, and the ER resident IP(3)R1 from brain microsomal extracts. Antibodies against the various signaling and Ca(2+) transport proteins coimmunoprecipitate Sec8 and the other signaling proteins. Dissociation of actin filaments in the immunoprecipitate had no effect on the interaction between Sec6 and Sec8, but released the actin and dissociated the interaction between the Sec6/8 complex and Ca(2+) signaling proteins. Hence, the interaction between the Sec6/8 and Ca(2+) signaling complexes is likely mediated by the actin cytoskeleton. The anti-Sec6 and anti-Sec8 antibodies inhibited Ca(2+) signaling at a step upstream of Ca(2+) release by IP(3). Disruption of the actin cytoskeleton with latrunculin B in intact cells resulted in partial translocation of Sec6 and Sec8 from membranes to the cytosol and interfered with propagation of agonist-evoked Ca(2+) waves. Our results suggest that the Sec6/8 complex has multiple roles in secretory cells including governing the polarized expression of Ca(2+) signaling complexes and regulation of their activity.     

ADP ribosylation factor-like protein 2 (Arl2) regulates the interaction of tubulin-folding cofactor D with native tubulin

The ADP ribosylation factor-like proteins (Arls) are a family of small monomeric G proteins of unknown function. Here, we show that Arl2 interacts with the tubulin-specific chaperone protein known as cofactor D. Cofactors C, D, and E assemble the alpha/beta- tubulin heterodimer and also interact with native tubulin, stimulating it to hydrolyze GTP and thus acting together as a beta-tubulin GTPase activating protein (GAP). We find that Arl2 downregulates the tubulin GAP activity of C, D, and E, and inhibits the binding of D to native tubulin in vitro. We also find that overexpression of cofactors D or E in cultured cells results in the destruction of the tubulin heterodimer and of microtubules. Arl2 specifically prevents destruction of tubulin and microtubules by cofactor D, but not by cofactor E. We generated mutant forms of Arl2 based on the known properties of classical Ras-family mutations. Experiments using these altered forms of Arl2 in vitro and in vivo demonstrate that it is GDP-bound Arl2 that interacts with cofactor D, thereby averting tubulin and microtubule destruction. These data establish a role for Arl2 in modulating the interaction of tubulin-folding cofactors with native tubulin in vivo.     

Tau Protein Function in Axonal Formation

Tau protein is a predominantly neuronal microtubule-associated protein that is enriched in axons and is capable of promoting microtubule assembly and stabilization. In the present article we review some of the key experiments directed to obtain insights about tau protein function in developing neurons. Aspects related to whether or not tau has essential, unique, or complementary functions during axonal formation are discussed.     

Tensile Tissue Stress Affects the Orientation of Cortical Microtubules in the Epidermis of Sunflower Hypocotyl

Abstract In turgid multicellular organs, it is convenient to differentiate between the two kinds of tensile forces acting in cell walls as a result of turgor pressure. The primary forces occur both in situ and in cells isolated from the organ, whereas the secondary forces occur only in situ. The latter are an unavoidable physical consequence of the variation in mechanical parameters of tissues forming layers or strands. The most rigid tissue is under maximal tensile force, whereas the least rigid is under maximal compressive force. These forces cause tissue stresses (that is, certain tissues are under tensile stress, whereas others are under compressive stress in the organ). The primary and secondary forces result in primary and secondary stress in cell walls, respectively. The anisotropy of the primary stress is a function of cell shape. For instance, in cylindric cells the anisotropy expressed as the ratio of longitudinal to transverse stresses is 0.5. The anisotropy of the secondary stress is a function of the compound structure of the organ. For example, in the epidermis of sunflower hypocotyl, the longitudinal secondary stress is much higher than the transverse stress. The primary and secondary stresses are superimposed, and, as a consequence, the stress anisotropy in the outer thick walls of epidermal cells is greater than 1. These outer epidermal walls transmit most of the tissue stress. When the epidermis is peeled but remains turgid, only primary stress remains, but loading of the peel can reestablish the original stress anisotropy. We studied the effect of stress anisotropy changes on the orientation of cortical microtubules (CMTs) in the sunflower hypocotyl epidermis. We showed that changes in stress anisotropy cause the CMT orientation to change in the direction of maximal wall stress. In situ, the relatively high tensile tissue stress in the epidermis causes maximal stress in the longitudinal direction and relatively steep CMT orientation. When the tissue stress is removed from the epidermis by peeling, the CMTs tend to reorient toward the transverse direction, which is the direction of maximal stress in the primary component. On application of external longitudinal stress, to substitute for tissue stress, CMTs tend to reorient in the longitudinal direction. However, a relatively high rate of plastic strain is caused by the stress applied to the peel in an acid medium. This produces a less steep orientation of CMTs. It appears that the change in stress anisotropy orients the CMT in the direction in which the stress is maximal after the change, but there is also some effect of the growth rate on the orientation. Received 4 January 2000; accepted 10 February, 2000     

Radial spindle and the phenotype of the maize meiotic mutant, dv

The morphological phenotype of the maize meiotic mutant dv (divergent spindle) has been further analysed by visualization of the division spindle and examination of its fine structure in mother cells of pollen. Previous research showed that dv blocks convergence of spindle fibres at the poles. New observations reveal abnormalities caused by this mutation, with dv showing disturbances in nuclear envelope breakdown during vesiculation, preventing the spindle fibres from adopting a bipolar orientation (with convergence on the poles). The anomalies result in radial spindles which are similar to monoastral spindles in animal cells.     

Structural and functional differences between porcine brain and budding yeast microtubules

The cytoskeleton of eukaryotic cells relies on microtubules to perform many essential functions. We have previously shown that, in spite of the overall conservation in sequence and structure of tubulin subunits across species, there are differences between mammalian and budding yeast microtubules with likely functional consequences for the cell. Here we expand our structural and function comparison of yeast and porcine microtubules to show different distribution of protofilament number in microtubules assembled in vitro from these two species. The different geometry at lateral contacts between protofilaments is likely due to a more polar interface in yeast. We also find that yeast tubulin forms longer and less curved oligomers in solution, suggesting stronger tubulin:tubulin interactions along the protofilament. Finally, we observed species-specific plus-end tracking activity for EB proteins: yeast Bim1 tracked yeast but not mammalian MTs, and human EB1 tracked mammalian but not yeast MTs. These findings further demonstrate that subtle sequence differences in tubulin sequence can have significant structural and functional consequences in microtubule structure and behavior.     

Actin filaments and microtubules in dendritic spines

Dendritic spines are small protrusions emerging from their parent dendrites, and their morphological changes are involved in synaptic plasticity. These tiny structures are composed of thousands of different proteins belonging to several subfamilies such as membrane receptors, scaffold proteins, signal transduction proteins, and cytoskeletal proteins. Actin filaments in dendritic spines consist of double helix of actin protomers decorated with drebrin and ADF/cofilin, and the balance of the two is closely related to the actin dynamics, which may govern morphological and functional synaptic plasticity. During development, the accumulation of drebrin‐binding type actin filaments is one of the initial events occurring at the nascent excitatory postsynaptic site, and plays a pivotal role in spine formation as well as small GTPases. It has been recently reported that microtubules transiently appear in dendritic spines in correlation with synaptic activity. Interestingly, it is suggested that microtubule dynamics might couple with actin dynamics. In this review, we will summarize the contribution of both actin filaments and microtubules to the formation and regulation of dendritic spines, and further discuss the role of cytoskeletal deregulation in neurological disorders.     

The distribution of TPX2 in dividing leaf cells of the fern Asplenium nidus

Plant cell division requires the dynamic organisation of several microtubule arrays. The mechanisms of regulation of the above arrays are under rigorous research. Among several factors that are involved in plant microtubule dynamics, the Targeting Protein for Xklp2 (TPX2) has been found to play a role in spindle organisation, in combination with Aurora kinases, in dividing cells of angiosperms. Microtubule organisation in dividing cells of ferns exhibits certain peculiarities. Accordingly, the presence and distribution of a TPX2 homologue might be helpful in understanding the patterns and regulatory mechanisms of microtubule arrays in this plant group. In this study, a putative TPX2 homologue was identified using Western blotting in the fern Asplenium nidus. It was found, using immunostaining and CLSM, that it is co‐localised with perinuclear preprophase microtubules and the prophase spindle, and follows the microtubule pattern during metaphase/anaphase and telophase. During cytokinesis, while in angiosperms TPX2 is degraded, in A. nidus the TPX2 signal persists, co‐localising with the phragmoplast. In early post‐cytokinetic cells, a TPX2 signal is present on the nuclear surface facing the daughter cell wall and, thereafter it is co‐localised with the fern‐specific microtubule aggregation that lines the new wall, which is possibly involved in cortical microtubule assembly.