Figure 3.

Centrosome-associated clathrin is membrane bound and distinct from the spindle-associated pool. A) Metaphase NRK cells were either untreated (a, b, e, f) or treated with 30 μM nocodazole (NZ) for 90 min (c, d, g, h). Some cells were also treated with 0.1% TritonX-100 (e–h). All cells were prepared for immunofluorescence microscopy as described previously (25). B) Centrosomes were prepared from 293T cells in the presence (+) or absence (−) of NP-40 and analyzed by Western blotting. Note that cytoskeletal components were disrupted prior to cell lysis by treatment with cytochalasin B and nocodazole. Amount of CHC isolated in the centrosome prep was quantified by densitometric analysis. C) Interphase (a–c) and mitotically synchronized (prometaphase, d–f; telophase, g–i) NRK cells were treated with nocodazole, followed by treatment with 0.1% TritonX-100, and probed with antibodies against clathrin (a, d, g) and γ-tubulin (b, e, h) for immunofluorescence microscopy; merge panels show colocalizaton (c, f, i). As a result of TX-100 treatment, CHC structures are displaced from all cellular compartments where they normally localize. Scale bars = 10 μm.