Kif18B is a newly discovered plus-tip-tracking protein that is enriched on astral microtubule (MT) ends during early mitosis. Kif18B binds directly to EB1, and this interaction is required for proper localization of Kif18B and to control astral MT length.

Summary The mitotic spindle is a macromolecular structure utilized to properly align and segregate sister chromatids to two daughter cells. During mitosis the spindle maintains a constant length, even though the spindle microtubules (MTs) are constantly undergoing polymerization and depolymerization [1]. Members of the Kinesin-8 family are important for the regulation of spindle length and for chromosome positioning [2–9]. Kinesin-8 proteins are length specific, plus-end directed motors that are proposed to be either MT depolymerases [3, 4, 8, 10, 11] or MT capping proteins [12]. How Kif18A uses its destabilization activity to control spindle morphology is not known. We found that Kif18A controls spindle length independently of its role in chromosome positioning. The ability of Kif18A to control spindle length is mediated by an ATP-independent MT binding site at the C-terminal end of the Kif18A tail that has a strong affinity for MTs in vitro and in cells. We used computational modeling to ask how modulating the motility or binding properties of Kif18A would affect its activity. Our modeling predicts that both fast motility and a low off-rate from the MT end are important for Kif18A function. In addition our studies provide new insight into how depolymerizing and capping enzymes can lead to MT destabilization.

The transmembrane proteins Delta and Notch act as ligand and receptor in a conserved signaling pathway required for a variety of cell fate specification events in many organisms. Binding of Delta to Notch results in a proteolytic cascade that releases the Notch intracellular domain, allowing it to participate in transcriptional activation in the nucleus. Recent research has implicated the endocytic and ubiquitylation machinery as essential components of Delta-Notch signaling. Our analysis of chimeric and missense Delta variants has delineated a number of structural requirements for Delta trafficking, receptor binding, and signaling. We find that while the Delta N-terminal domain is necessary and sufficient for binding to Notch, the integrity of the epidermal-growth-factor-l ike repeat (ELR) 2 is also required for Notch binding. Screening of 117 Delta mutant lines for proteins that exhibit aberrant subcellular trafficking has led to the identification of 18 Delta alleles (Dl TD alleles) that encode ''trafficking-defective'' Delta proteins. We find, unexpectedly, that many Dl TD alleles contain missense mutations in ELRs within the Delta extracellular domain. Finally, we find that two Dl TD alleles contain lysine missense mutations within the Delta intracellular domain (DeltaICD) that may identify residues important for DeltaICD mono-ubiquitylation and subsequent Delta endocytosis and signaling.

Endocytosis of the ligand delta; is required for activation of the receptor Notch during Drosophila development. The Notch extracellular domain (NotchECD) dissociates from the Notch intracellular domain (NotchICD) and is trans-endocytosed into delta;-expressing cells in wild-type imaginal discs. Reduction of dynamin-mediated endocytosis in developing eye and wing imaginal discs reduces Notch dissociation and Notch signalling. Furthermore, dynamin-mediated delta endocytosis is required for Notch trans-endocytosis in Drosophila cultured cell lines. Endocytosis-defective delta proteins fail to mediate trans-endocytosis of Notch in cultured cells, and exhibit aberrant subcellular trafficking and reduced signalling capacity in Drosophila. We suggest that endocytosis into delta-expressing cells of NotchECD bound to delta plays a critical role during activation of the Notch receptor and is required to achieve processing and dissociation of the Notch protein.

Disorazoles comprise a family of 29 macrocyclic polyketides isolated from the fermentation broth of the myxobacterium Sorangium cellulosum. The major fermentation product, disorazole A 1 , was found previously to irreversibly bind to tubulin and to have potent cytotoxic activity against tumor cells, possibly because of its highly electrophilic epoxide moiety. To test this hypothesis, we synthesized the epoxide-free disorazole C 1 and found it retained potent antiproliferative activity against tumor cells, causing prominent G 2 /M phase arrest and inhibition of in vitro tubulin polymerization. Furthermore, disorazole C 1 produced disorganized microtubules at interphase, misaligned chromosomes during mitosis, apoptosis, and premature senescence in the surviving cell populations. Using a tubulin polymerization assay, we found disorazole C 1 inhibited purified bovine tubulin polymerization, with an IC 50 of 11.8 Ϯ 0.4 M, and inhibited [ 3 H]vinblastine binding noncompetitively, with a K i of 4.5 Ϯ 0.6 M. We also found noncompetitive inhibition of [ 3 H]dolastatin 10 binding by disorazole C 1 , with a K i of 10.6 Ϯ 1.5 M, indicating that disorazole C 1 bound tubulin uniquely among known antimitotic agents. Disorazole C 1 could be a valuable chemical probe for studying the process of mitotic spindle disruption and its relationship to premature senescence.

Spatial and temporal control of MT dynamics is important for proper spindle assembly and chromosome segregation. The kinesin-8 Kif18B spatially regulates astral MT dynamics. Not all members of a single kinesin superfamily control MT dynamics in a similar fashion.

Dynamic microtubules are necessary for proper mitotic spindle assembly and chromosome segregation during mitosis. Members of the kinesin superfamily of molecular motor proteins are important to spindle function. Of particular interest is the Kinesin-13 family member MCAK, which acts to regulate microtubule dynamics during spindle assembly and to ensure proper attachments of chromosomes to spindle microtubules. The unique ability of MCAK to regulate microtubule dynamics makes it a potential target for development of new drugs that alter spindle function. Here, we knocked down MCAK via RNAi in normal and malignant cell lines and found that the two tested malignant cell lines were acutely sensitive to MCAK knockdown, while the tested normal cells were less sensitive. In addition, we looked at the effect of combining MCAK knockdown and drug treatment with paclitaxel or vinblastine to identify spindle assembly defects. We found that MCAK knockdown increased the morphological defects of the microtubule cytoskeleton in HeLa cells caused by anti-microtubule drugs. Our studies support the idea that MCAK would be a good target for new chemotherapeutic development and may be particularly useful in combination therapies with currently available anti-microtubule agents.

A model for generating highly polyploid cells through endoreplication shows that iECs have lower levels of HSET, overexpression of which rescues spindle multipolarity. The results provide insight into the mechanisms by which alternative cell cycles lead to polyploid cell division and genomic instability.