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First published online 14 November 2007
doi: 10.1242/jcs.012468

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In vivo movement of the type V myosin Myo52 requires dimerisation but is independent of the neck domain

¹ Cancer Research UK Cell Division Group, CR-UK Paterson Institute for Cancer Research, Manchester, M20 4BX, UK
² Cell and Developmental Biology Group, Department of Biosciences, University of Kent, Canterbury, Kent, CT2 7NJ, UK
³ Advanced imaging facility, CR-UK Paterson Institute for Cancer Research, Manchester, M20 4BX, UK

View larger version (56K): [in this window] [in a new window]   Fig. 1. In vivo analysis of Myo52 movement. (a) Live timelapse analysis of the Myo52-cGFP strain revealed slow short movements (yellow and green arrows) and fast long directed movements (red, white and blue arrows). Myo52 foci are often seen to change poleward direction of travel (white arrow). (b,c) Cells expressing either Myo52-nGFP or GFP-Atb2 (arrows) were mixed and Myo52 movements were monitored in the (b) absence or (c) presence of 25 µg/ml carbendazim. Microtubule depolymerisation did not affect Myo52 movement. (d-f) Myo52 movements and the actin-patch movement were monitored simultaneously in cells expressing either Myo52-nGFP or Crn1-GFP (arrows) when they were incubated in the presence of (d) DMSO, (e) 2 µM or (f) 20 µM latrunculin A. (g-j) Kymographs of 100x100 msecond timelapse frames of Myo52-cGFP cells treated with (g) DMSO, (h) carbendazim, (i) 2 µM or (j) 20 µM latrunculin A demonstrate that rapid long-distance Myo52 movements are not affected by microtubule depolymerisation but abolished in the absence of actin cables. Bars, 10 µm.

View larger version (60K): [in this window] [in a new window]   Fig. 2. Myo52 movement upon actin filaments is driven by its own motor activity drives. (a,b) Myo52-cGFP localisation (middle panels, red labelling in bottom panels) is abolished in mutant cells (arrows) that lack (a) the fission yeast tropomyosin or (b) profillin, but not in simultaneously observed TRITC-lectin-labelled wild-type cells (upper panels, green labelling in bottom panels). (c) Myo52 links with medial-ring-associated actin filaments in for3 cells, lacking the normal interphase filament distribution. (d) Velocity distributions of Myo52 foci (green) and GFP-Crn1 labelled actin patches (red) are significantly different. (e,f) ATP depletion using 10 nM FCCP abolishes (f) Myo52 but not (e) actin-patch movement. (g) Point mutations introduced to residues within the Myo52 protein that were predicted to disrupt the motor function of the protein. Growth curves indicate that none of the mutant proteins were able to complement the myo52 allele. (h) Compared with wild-type Myo52 movements of these rigour mutants had a reduced velocity (Y509G and G689A) or were abolished completely (F490A and G689V).

View larger version (79K): [in this window] [in a new window]   Fig. 3. Myo52 function and movement are not dependent upon its neck region. (a) Myo52 protein with boxed IQ-motif sequences. Residues deleted from GFP-Myo52IQ are underlined. (b) Yeast two-hybrid assay showing interaction of the Myo52 tail with itself or the fission yeast calmodulin Cam1. (c) Anti-GFP western blot of myo52-strain extracts expressing Myo52-nGFP, GFP-Myo52IQ or GFP-Myo52CC confirm the expected reduced size of the Myo52 mutant in the latter strain. (d,e) Micrographs of (d) Myo52CC and (e) Myo52IQ cells show that only Myo52IQ localised normally and was able to complement the morphology defect associated with the myo52 allele. (f) Kymographs of Myo52IQ cells demonstrate that the truncation mutant can make rapid directed and bidirectional movements throughout the cell. (g) Velocity distributions of Myo52 and Myo52IQ. Bars, 10 µm.

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