In all eukaryotes, a microtubule-based structure known as the spindle is responsible for accurate chromosome segregation during cell division. Spindle assembly and function require localized regulation of microtubule dynamics and the activity of a variety of microtubule-based motor proteins. Recent work has begun to uncover the molecular mechanisms that underpin this process. Here we describe the structural and dynamic properties of the spindle, and introduce the current concepts regarding how a bipolar spindle is assembled and how it functions to segregate chromosomes.

TPX2, the targeting protein for Xenopus kinesin-like protein 2 (Xklp2), was identified as a microtubule-associated protein that mediates the binding of the COOH-terminal domain of Xklp2 to microtubules (Wittmann, T., H. Boleti, C. Antony, E. Karsenti, and I. Vernos. 1998. J. Cell Biol. 143:673–685). Here, we report the cloning and functional characterization of Xenopus TPX2. TPX2 is a novel, basic 82.4-kD protein that is phosphorylated during mitosis in a microtubule-dependent way. TPX2 is nuclear during interphase and becomes localized to spindle poles in mitosis. Spindle pole localization of TPX2 requires the activity of the dynein–dynactin complex. In late anaphase TPX2 becomes relocalized from the spindle poles to the midbody. TPX2 is highly homologous to a human protein of unknown function and thus defines a new family of vertebrate spindle pole components. We investigated the function of TPX2 using spindle assembly in Xenopus egg extracts. Immunodepletion of TPX2 from mitotic egg extracts resulted in bipolar structures with disintegrating poles and a decreased microtubule density. Addition of an excess of TPX2 to spindle assembly reactions gave rise to monopolar structures with abnormally enlarged poles. We conclude that, in addition to its function in targeting Xklp2 to microtubule minus ends during mitosis, TPX2 also participates in the organization of spindle poles.

The obligate intracellular pathogen Chlamydia trachomatis replicates within a membrane-bound inclusion that acquires host sphingomyelin (SM), a process that is essential for replication as well as inclusion biogenesis. Previous studies demonstrate that SM is acquired by a Brefeldin A (BFA)-sensitive vesicular trafficking pathway, although paradoxically, this pathway is dispensable for bacterial replication. This finding suggests that other lipid transport mechanisms are involved in the acquisition of host SM. In this work, we interrogated the role of specific components of BFA-sensitive and BFA-insensitive lipid trafficking pathways to define their contribution in SM acquisition during infection. We found that C. trachomatis hijacks components of both vesicular and non-vesicular lipid trafficking pathways for SM acquisition but that the SM obtained from these separate pathways is being utilized by the pathogen in different ways. We show that C. trachomatis selectively co-opts only one of the three known BFA targets, GBF1, a regulator of Arf1-dependent vesicular trafficking within the early secretory pathway for vesicle-mediated SM acquisition. The Arf1/GBF1-dependent pathway of SM acquisition is essential for inclusion membrane growth and stability but is not required for bacterial replication. In contrast, we show that C. trachomatis co-opts CERT, a lipid transfer protein that is a key component in non-vesicular ER to trans-Golgi trafficking of ceramide (the precursor for SM), for C. trachomatis replication. We demonstrate that C. trachomatis recruits CERT, its ER binding partner, VAP-A, and SM synthases, SMS1 and SMS2, to the inclusion and propose that these proteins establish an on-site SM biosynthetic factory at or near the inclusion. We hypothesize that SM acquired by CERT-dependent transport of ceramide and subsequent conversion to SM is necessary for C. trachomatis replication whereas SM acquired by the GBF1-dependent pathway is essential for inclusion growth and stability. Our results reveal a novel mechanism by which an intracellular pathogen redirects SM biosynthesis to its replicative niche.

Regulation of microtubule dynamics is essential for many cell biological processes, and is likely to be variable between different subcellular regions. We describe a computational approach to analyze microtubule dynamics by detecting growing microtubule plus ends. Our algorithm tracks all EB1-EGFP comets visible in an image time-lapse sequence allowing the detection of spatial patterns of microtubule dynamics. We use spatiotemporal clustering of EB1-EGFP growth tracks to infer microtubule behaviors during phases of pause and shortening. The algorithm was validated by comparison to manually tracked, homogeneously labeled microtubules, and by analysis of the effects of well-characterized inhibitors of microtubule polymerization dynamics. We used our method to analyze spatial variations of intracellular microtubule dynamics in migrating epithelial cells.

Turnover of integrin-based focal adhesions (FAs) with the extracellular matrix (ECM) is essential for coordinated cell movement. In collectively migrating human keratinocytes, FAs assemble near the leading edge, grow and mature as a result of contractile forces, and disassemble underneath the advancing cell body. We report that clustering of microtubule-associated CLASP1 and CLASP2 proteins around FAs temporally correlates with FA turnover. CLASPs and LL5β, which recruits CLASPs to FAs, facilitate FA disassembly. CLASPs are further required for FA-associated ECM degradation, and matrix metalloprotease inhibition slows FA disassembly similar to CLASP or LL5β depletion. Finally, CLASP-mediated microtubuletethering at FAs establishes a FA-directed transport pathway for delivery, docking and localized fusion of exocytic vesicles near FAs. We propose that CLASPs couple microtubule organization, vesicle transport and cell interactions with the ECM, establishing a local secretion pathway that facilitates FA turnover by severing cell-matrix connections.