The interplay of the N- and C-terminal domains of MCAK control microtubule depolymerization activity and spindle assembly.

Spindle assembly and accurate chromosome segregation require the proper regulation of microtubule dynamics. MCAK, a Kinesin-13, catalytically depolymerizes microtubules, regulates physiological microtubule dynamics, and is the major catastrophe factor in egg extracts. Purified GFP-tagged MCAK domain mutants were assayed to address how the different MCAK domains contribute to in vitro microtubule depolymerization activity and physiological spindle assembly activity in egg extracts. Our biochemical results demonstrate that both the neck and the C-terminal domain are necessary for robust in vitro microtubule depolymerization activity. In particular, the neck is essential for microtubule end binding, and the C-terminal domain is essential for tight microtubule binding in the presence of excess tubulin heterodimer. Our physiological results illustrate that the N-terminal domain is essential for regulating microtubule dynamics, stimulating spindle bipolarity, and kinetochore targeting; whereas the C-terminal domain is necessary for robust microtubule depolymerization activity, limiting spindle bipolarity, and enhancing kinetochore targeting. Unexpectedly, robust MCAK microtubule (MT) depolymerization activity is not needed for sperm-induced spindle assembly. However, high activity is necessary for proper physiological MT dynamics as assayed by Ran-induced aster assembly. We propose that MCAK activity is spatially controlled by an interplay between the N- and C-terminal domains during spindle assembly.

Tetracycline labeling as a measure of humeral head viability after 3- or 4-part proximal humerus fracture.

HYPOTHESIS: Specifically located humeral head biopsies from three-part proximal humerus fractures taken at the time of hemiarthroplasty will show greater tetracycline labeling under fluorescent microscopy than those biopsies taken from four-part fractures. Additionally, biopsies from younger patients would show greater fluorescence than older patients. MATERIAL AND METHODS: Nineteen consecutive adult patients (range 43-83 years) underwent hemiarthroplasty as definitive treatment for 20 displaced three- and four-part proximal humerus fractures after having received 500 mg of tetracycline hydrochloride orally every six hours for the immediate five preoperative days. Humeral head biopsies were taken from four pre-determined locations intraoperatively. The biopsies were prepared and analyzed with fluorescent microscopy. RESULTS: All specimens in each biopsy location demonstrated fluorescence. There was no difference between the mean rank gray values for the four biopsy locations (p = 0.78 with the Friedman test). There was no difference between the mean rank gray values for the four biopsy locations when analyzed according to three-part vs four-part fracture (p > 0.05 with the Mann-Whitney test). There was an inverse relationship between age and fluorescence for the anterosuperior biopsy location (p = 0.033 with Spearman correlation). DISCUSSION: Vascular supply is preserved in displaced three- and four-part proximal humerus fractures. With intact vascularity to the humeral head, head-preserving techniques utilizing stable, site-specific fixation and minimal dissection should be considered in the treatment of displaced three- and four-part proximal humerus fractures. CONCLUSION: Vascular supply is preserved in displaced three- and four-part proximal humerus fractures, especially in younger patients in the anterosuperior aspect of the humeral head. LEVEL OF EVIDENCE: Basic Science Study.

The uncovering of a novel regulatory mechanism for PLD2: formation of a ternary complex with protein tyrosine phosphatase PTP1B and growth factor receptor-bound protein GRB2.

The regulation of PLD2 activation is poorly understood at present. Transient transfection of COS-7 with a mycPLD2 construct results in elevated levels of PLD2 enzymatic activity and tyrosyl phosphorylation. To investigate whether this phosphorylation affects PLD2 enzymatic activity, anti-myc immunoprecipitates were treated with recombinant protein tyrosine phosphatase PTP1B. Surprisingly, lipase activity and PY levels both increased over a range of PTP1B concentrations. These increases occurred in parallel to a measurable PTP1B-associated phosphatase activity. Inhibitor studies demonstrated that an EGF-receptor type kinase is involved in phosphorylation. In a COS-7 cell line created in the laboratory that stably expressed myc-PLD2, PTP1B induced a robust (>6-fold) augmentation of myc-PLD2 phosphotyrosine content. The addition of growth factor receptor-bound protein 2 (Grb2) to cell extracts also elevated PY levels of myc-PLD (>10-fold). Systematic co-immunoprecipitation-immunoblotting experiments pointed at a physical association between PLD2, Grb2, and PTP1B in both physiological conditions and in overexpressed cells. This is the first report of a demonstration of the mammalian isoform PLD2 existing in a ternary complex with a protein tyrosine phosphatase, PTP1b, and the docking protein Grb2 which greatly enhances tyrosyl phosphorylation of the lipase.