Motility of Single Molecules and Clusters of Bi-Directional Kinesin-5 Cin8 Purified from S. cerevisiae Cells.

The mitotic bipolar kinesin-5 motors perform essential functions in spindle dynamics. These motors exhibit a homo-tetrameric structure with two pairs of catalytic motor domains, located at opposite ends of the active complex. This unique architecture enables kinesin-5 motors to crosslink and slide apart antiparallel spindle microtubules (MTs), thus providing the outwardly-directed force that separates the spindle poles apart. Previously, kinesin-5 motors were believed to be exclusively plus-end directed. However, recent studies revealed that several fungal kinesin-5 motors are minus-end directed at the single-molecule level and can switch directionality under various experimental conditions. The Saccharomyces cerevisiae kinesin-5 Cin8 is an example of such bi-directional motor protein: in high ionic strength conditions single molecules of Cin8 move in the minus-end direction of the MTs. It was also shown that Cin8 forms motile clusters, predominantly at the minus-end of the MTs, and such clustering allows Cin8 to switch directionality and undergo slow, plus-end directed motility. This article provides a detailed protocol for all steps of working with GFP-tagged kinesin-5 Cin8, from protein overexpression in S. cerevisiae cells and its purification to in vitro single-molecule motility assay. A newly developed method described here helps to differentiate between single molecules and clusters of Cin8, based on their fluorescence intensity. This method enables separate analysis of motility of single molecules and clusters of Cin8, thus providing the characterization of the dependence of Cin8 motility on its cluster size.

Incidental finding of hereditary multiple osteochondroma causing ischiofemoral impingement.

Ischiofemoral impingement (IFI) has been described in the medical literature as a cause of hip pain. IFI occurs due to an abnormal contact or reduced space between the lesser trochanter and the lateral border of the ischium and is an often unrecognised cause of pain and snapping in the hip. Association of multiple exostoses and a skeletal dysplasia characterised by an abnormal modelling of bone metaphysis and osseous deformities is highly characteristic of this disease. Consequently, multiple exostoses may narrow the ischiofemoral space and cause impingement and pain, even in the absence of malignant transformation. Surgical excision of exostosis of the lesser trochanter is a safe and effective method of treatment for patients with IFI. We present a case of left hip pain with incidental finding of hereditary multiple osteochondroma causing IFI and discuss the predisposing factors and review of literature.

An anatomical study of tibial metaphyseal/diaphyseal mismatch during revision total knee arthroplasty.

This cadaveric study determines the relationship of the tibial metaphysis and intramedullary canal of the tibia (diaphysis), particularly relating to revision knee arthroplasty. A total knee arthroplasty was performed in 20 cadaveric tibiae using revision instrumentation with a canal-filling uncemented stem. The offset, which is the distance between the center of the tibial canal and the center of the tibial metaphysis, was measured using the revision system offset bushing. The average distance between the center of the tibial metaphysis and the center of the tibial diaphysis was 4.1 mm (+/-2.9 mm) with an average angle of 105 degrees (0 degrees -70 degrees ). The mean offset was 4.1 mm (+/-2.9) and the mean offset angle was 105 degrees (0 degrees -350 degrees ). The study confirms the center of the tibial diaphysis is not congruous with the center of the metaphysis with wide variations in offsets among individuals. A wide range of offsets are necessary for optimum placement of implants.