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. 2002 Jun;13(6):1832–1845. doi: 10.1091/mbc.01-10-0504

Figure 3.

Figure 3

ATP-binding mutant of CHO1 (CHO1F′) interacts with microtubules via ATP-independent microtubule binding site(s) but does not concentrate at the midline of the central spindle during anaphase. (A) CHO1M associates with microtubules when expressed in CHO cells. (B) CHO1 M′ generally does not display microtubule binding (compare HA staining in B and tubulin staining in B′). CHO1F′ interacts with microtubules in both mitotic (C, metaphase; D, anaphase; E, telophase) and interphase cells (F). Although CHO1F′ contains the NLS and is generally seen within the nucleus, in some interphase cells with the high level of expression, CHO1F′ can also be detected in the cytoplasm (F). CHO1F′ΔT strongly binds and bundles microtubules (G), whereas endogenous CHO1 is confined within the nucleus (G′). This indicates that microtubule binding by mutated CHO1F′ and CHO1F′ΔT constructs is not due to the dimerization with endogenous CHO1 but is due to the ATP-independent microtubule binding site(s) present in the motor protein. CHO1F′ fails to concentrate at the midline of the central spindle during anaphase (D) and distributes along the entire intercellular bridge at the end of cytokinesis (E). Staining was performed using monoclonal anti-HA (A and C–E), polyclonal anti-HA (B, F, and G), monoclonal antitubulin (B′ and F′), monoclonal anti-CHO1 antibodies (G′) and DAPI (F" and G"). Bars, 10 μm.