Microtubule cytoskeleton: navigating the intracellular landscape

Recent studies have significantly advanced our understanding of how a dividing cell asymmetrically positions the mitotic spindle--a key process in metazoan development--while maintaining a dynamic spindle state that can respond and reorient when necessary.     

Microtubule assembly dynamics: new insights at the nanoscale

Although the dynamic self-assembly behavior of microtubule ends has been well characterized at the spatial resolution of light microscopy (~200 nm), the single-molecule events that lead to these dynamics are less clear. Recently, a number of in vitro studies used novel approaches combining laser tweezers, microfabricated chambers, and high-resolution tracking of microtubule-bound beads to characterize mechanochemical aspects of MT dynamics at nanometer scale resolution. In addition, computational modeling is providing a framework for integrating these experimental results into physically plausible models of molecular scale microtubule dynamics. These nanoscale studies are providing new fundamental insights about microtubule assembly, and will be important for advancing our understanding of how microtubule dynamic instability is regulated in vivo via microtubule-associated proteins, therapeutic agents, and mechanical forces.     

Microtubule cytoskeleton: No longer an also Ran.

The Ran GTPase cycle has been extensively studied in the context of nuclear transport. Recent work indicates that this GTPase cycle also plays an important role in regulating the microtubule cytoskeleton.     

Microtubule dynamics: Patronin, protector of the minus end

It has long been surmised that cellular microtubules are capped at the minus ends to prevent their depolymerization. A recent study provides the first definitive identification of a minus-end-specific capping protein, termed Patronin, which protects the microtubule arrays of both mitotic and interphase cells.     

Microtubule end binding: EBs sense the guanine nucleotide state

EB proteins accumulate at the tips of growing microtubules and recruit to them a multitude of factors to regulate microtubule functions. A new study suggests that EBs recognize microtubule ends by distinguishing between different states of the tubulin-bound guanine nucleotide.     

Microtubule dynamics and signal transduction at the immunological synapse: new partners and new connections

EMBO J (2012) 31 21, 4140–4152 doi:10.1038/emboj.2012.242; published online August242012Antigen recognition induces T cells to polarize towards antigen presenting cells (APC) generating an organized cell interface named the immunological synapse. T-cell microtubules (MTs) reorient the MT-organizing centre (MTOC) to the immunological synapse central region, while MT irradiate towards the synapse periphery. Martín-Cófreces et al (2012) describe in this issue that the MT plus-end-binding protein 1 (EB1) interacts with TCR cytosolic regions and mediate the organization of an immunological synapse fully functional to transduce activation signals.The pioneer work of Kupfer and Singer (1989) established that T-cell MTs rearrange in response to specific TCR engagement by APCs, resulting in MTOC orientation to the APC contact site in helper and cytotoxic T cells. MTOC reorientation was shown to be the result of a MT polymerization dynamic process involving MT posttranslational modifications (Kuhn and Poenie, 2002; Serrador et al, 2004). MT reorganization during T-cell antigen recognition is functionally linked to T-cell effector functions, like the polarized secretion of helper cytokines to B cells (Kupfer et al, 1991; Huse et al, 2006), or cytotoxic granules to target cells (Stinchcombe et al, 2006). MTs also transport TCR-carrying endosomes during synapse formation (Das et al, 2004) and TCR signalling complexes at the immunological synapse (Lasserre et al, 2010; Hashimoto-Tane et al, 2011). Altogether, these findings show that the dynamic reorganization of MTs and its related molecular transport are critical for the organization and function of the immunological synapse.Martín-Cófreces et al (2012) present here interesting new insights, unveiling a link between EB1 and the TCR complex. EB1 is one of a series of MT plus-end-associated proteins critical for MT polymerization dynamics (Slep, 2010). The first important finding initially issued from a two-hybrid screening was that EB1 could directly interact with TCR complex cytosolic regions. By GST pull-down and co-immunoprecipitation experiments, the authors narrowed down this interaction to two of the TCR complex subunits, ζ and ɛ, in their ITAM (immuno-receptor tyrosine-based activation motif)-containing regions, and within the C-terminal 82 amino-acid region on EB1. In T cells, EB1–TCR interaction could occur without TCR stimulation, suggesting that EB1 plays a role in TCR dynamics previous to TCR engagement. The authors then investigated EB1 localization and its involvement in synapse organization and function. Live cell imaging showed intense EB1 movement in the synapse area, with MTs growing from the MTOC to the synapse periphery, leading to an apparent concentration of EB1 at the T cell–APC interface. To analyse the relationship between MT dynamics and intracellular transport, the authors followed EB1–GFP and TCRζ–Cherry by total internal reflection fluorescence (TIRF) microscopy in synapses formed on anti-CD3-coated cover slips. They observed transient coincident spots between EB1 and TCRζ+ vesicles, suggesting that growing MTs transport TCRζ-carrying vesicles towards the immunological synapse. Consistently, EB1-silenced cells displayed altered TCRζ vesicle dynamics and TCRζ clustering at the synapse. Likewise, vesicle transport to the synapse of the signalling scaffold molecule LAT and its clustering at the synapse were altered. Finally, they observed transient encounters between TCRζ- and LAT-carrying vesicles inhibited by EB1 silencing. These observations point out to a crucial role of EB1 and MT dynamics in the organization of the immunological synapse.Immunological synapse organization has been related with its capacity to regulate TCR signal transduction. Therefore, Martín-Cófreces et al (2012) investigated how EB1 silencing impacted TCR signalling. EB1-silenced cells were indeed impaired in key TCR signalling events, like LAT tyrosine phosphorylation, which allows LAT interaction with activation effectors, like the phospholipase C (PLC)γ, promoting TCR signal propagation. Consistently, PLCγ activation was impaired in EB1-silenced cells. However, upstream activation events, like tyrosine phosphorylation of TCRζ and of its associated protein tyrosine kinase ZAP70, were not altered. This suggests that MT-dependent LAT vesicle traffic is key for LAT phosphorylation and the generation of TCR signalling complexes.Altogether, Martín-Cófreces'' findings reinforce the idea that polarized vesicle transport via organized MT networks is key to set up the immunological synapse as a signal transduction platform. EB1 interaction with two TCR subunits may link the TCR complex with MTs dynamics. It remains unanswered, however, whether EB1 also interacts with LAT, facilitating the merging at the synapse of distinct TCRζ- and LAT-carrying vesicles.Vesicle traffic on MTs generally occurs via molecular motors from the dynein and kinesin families. The former are associated with minus end-oriented transport, whereas the later mostly ensures plus-end-associated transport. The immunological synapse may use both types of transport. Thus, cytotoxic granule delivery to the synapse may mainly involve dynein-mediated vesicle traffic, since the MTOC translocates very close to the immunological synapse (Stinchcombe et al, 2006). Likewise, centripetal movements of signalling microclusters at the synapse involve dynein (Hashimoto-Tane et al, 2011). Martín-Cófreces et al (2012) show that TCRζ- and LAT-carrying vesicles are transported towards MT plus ends in an EB1-dependent manner. It remains uncertain whether EB1 could play a direct transport role at the immunological synapse, helping the attachment of TCRζ vesicles to growing MT plus ends. Alternatively, EB1 could mediate MT interactions with TCR complexes present at the plasma membrane. Initial TCR clustering at the synapse would help capturing EB1-positive MT plus ends, orienting MTs and MT-mediated traffic of TCRζ- and LAT-carrying vesicles to the synapse by a kinesin-based transport (Figure 1), and promoting TCRζ and LAT encountering and clustering at the synapse. EB1 silencing would perturb MT–plasma membrane interactions impairing this MT orientation and transport loop. MT polymerization kinetic studies on immunological synapses formed by EB1-silenced versus control T cells may help to clarify this mechanism. Although further studies will be necessary to elucidate the detailed mechanism, the work by Martín-Cófreces et al (2012) already highlights the importance of MT dynamics and vesicle traffic in the formation of a functional immunological synapse, raising novel and interesting questions on how the MT network helps to set up complex signal transduction machineries.Open in a separate windowFigure 1Model of the role of EB1 in MT dynamics and TCR signal transduction at the immunological synapse. (A) Initial T cell–APC contact. TCR initial clustering would favour the capture of EB1-containing MT plus ends at the T cell–APC contact. (B) Immune synapse formation. The increase capture of MTs plus ends by TCR clusters would promote the arrival of TCRζ- and LAT-carrying vesicles leading to increase TCR and LAT clustering and encountering at the synapse. Alternatively, EB1 interaction with TCR could also be directly involved in TCRζ vesicle transport to the synapse. In turn, increase TCR clustering would promote additional MT and capture, building an amplification loop for MT dynamics and vesicle transport. (C) Established immunological synapse. A structured MT network would facilitate the continuous arrival of TCRζ- and LAT-carrying vesicles through the MT plus ends at the immunological synapse periphery. Then the centripetal movement of TCR signalling complexes towards the MT minus end at the MTOC close to the synapse centre would bring signalling complexes to signal extinction sites (i.e., endosomes). The right panel in C represents a xy section of the immunological synapse, as it is observed on stimulatory cover slips.     

Microtubule polymerization: one step at a time

The dynamic assembly of microtubules is a key factor in many of their functions in the cell and recent experiments give new insight into this process at the molecular level.     

Pattern formation: a new twist to BMP signalling

Dorsal-ventral patterning in Xenopus and Drosophila embryos involves BMP family signalling molecules. Twisted Gastrulation has now been added to the list of proteins that regulate the activity of these molecules, providing new insights into how BMPs are made available to their signalling receptors.     

Silk from crickets: a new twist on spinning

Raspy crickets (Orthoptera: Gryllacrididae) are unique among the orthopterans in producing silk, which is used to build shelters. This work studied the material composition and the fabrication of cricket silk for the first time. We examined silk-webs produced in captivity, which comprised cylindrical fibers and flat films. Spectra obtained from micro-Raman experiments indicated that the silk is composed of protein, primarily in a beta-sheet conformation, and that fibers and films are almost identical in terms of amino acid composition and secondary structure. The primary sequences of four silk proteins were identified through a mass spectrometry/cDNA library approach. The most abundant silk protein was large in size (300 and 220 kDa variants), rich in alanine, glycine and serine, and contained repetitive sequence motifs; these are features which are shared with several known beta-sheet forming silk proteins. Convergent evolution at the molecular level contrasts with development by crickets of a novel mechanism for silk fabrication. After secretion of cricket silk proteins by the labial glands they are fabricated into mature silk by the labium-hypopharynx, which is modified to allow the controlled formation of either fibers or films. Protein folding into beta-sheet structure during silk fabrication is not driven by shear forces, as is reported for other silks.     

Oxytropism: a new twist in pollen tube orientation

Chemical gradients and structural features within the pistil have been previously proposed as factors determining the directionality of pollen tube growth. In this study, we examine the behavior of pollen of eight species germinated in a dynamic oxygen gradient. While the germination rates of some species decreased directly with decreasing oxygen tension, other species showed no decrease in germination at oxygen tensions as low as 2 kPa. In one species, germination was consistently greater at decreased oxygen tensions than at ambient atmospheric levels. In three of the eight species tested, the developing pollen tube showed clear directional growth away from the more-oxygenated regions of the growth medium, while in one species growth was towards the more-oxygenated region. The remaining four species showed random tube growth. The pattern of oxytropic responses among the taxa suggests that this tropic behavior is both widespread and phylogenetically unpredictable.     

Torsional restraint: a new twist on frameshifting pseudoknots

mRNA pseudoknots have a stimulatory function in programmed −1 ribosomal frameshifting (−1 PRF). Though we previously presented a model for how mRNA pseudoknots might activate the mechanism for −1 PRF, it did not address the question of the role that they may play in positioning the mRNA relative to the ribosome in this process [E. P. Plant, K. L. M. Jacobs, J. W. Harger, A. Meskauskas, J. L. Jacobs, J. L. Baxter, A. N. Petrov and J. D. Dinman (2003) RNA, 9, 168–174]. A separate ‘torsional restraint’ model suggests that mRNA pseudoknots act to increase the fraction of ribosomes directed to pause with the upstream heptameric slippery site positioned at the ribosome's A- and P-decoding sites [J. D. Dinman (1995) Yeast, 11, 1115–1127]. Here, experiments using a series of ‘pseudo-pseudoknots’ having different degrees of rotational freedom were used to test this model. The results of this study support the mechanistic hypothesis that −1 ribosomal frameshifting is enhanced by torsional resistance of the mRNA pseudoknot.     

Tumor metastasis: a new twist on epithelial-mesenchymal transitions

Epithelial-mesenchymal transitions are essential for normal embryonic development and for progression of non-invasive tumor cells into malignant, metastatic carcinomas. Twist, an important regulator of morphogenesis in the embryo, has recently been implicated in the onset of invasive behavior during tumor progression.     

Microtubule cytoskeleton in intact and wounded coenocytic green algae

Microtubule (MT) arrangements were investigated, with immunofluorescence and electron microscopy, in two related species of coenocytic green algae. Intact cells of both Ernodesmis verticillata (Kützing) Boergesen and Boergesenia forbesii (Harvey) Feldmann have two morphologically distinct populations of MTs: a highly regular cortical array consisting of a single layer of parallel, longitudinal MTs; and perinuclear MTs radiating from the surface of the envelope of each interphase nucleus. In both algae, mitotic figures lack perinuclear MTs around them. Pre-incubation with taxol does not alter the appearance of these arrays. The cortical and nuclear MTs appear to coexist throughout the nuclear cycle, unlike the condition in most plant cells. At the cut/contracting ends of wounded Ernodesmis cells, cortical MTs exhibit bundling and marked convolution, with some curvature and slight bundling of MTs throughout the cell cortices. In Boergesenia, wound-induced reticulation and separation of the protoplasm into numerous spheres also involves a fasciation of MTs within the attenuating regions of the cytoplasm. Although some cortical MTs are fairly resistant to cold and amiprophos-methyl-induced depolymerization, the perinuclear ones are very labile, depolymerizing in 5–10 min in the cold. The MT cytoskeleton is not believed to be directly involved in wound-induced motility in these plants because amiprophos-methyl and cold depolymerize most cortical MTs without inhibiting motility. Also, the identical MT distributions in intact cells of these two algae belie the very different patterns of cytoplasmic motility. Although certain roles of the MT arrays may be ruled out, their exact functions in these plants are not known.Abbreviations APM amiprophos-methyl - DIC differential interference contrast - EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - FITC fluorescein isothiocyanate - MT(s) microtubule(s) - PBS phosphate-buffered saline