Calmodulin association with and stabilization of kinetochore microtubules of the mammalian mitotic spindle.

Abstract

We have investigated the extent and nature of the association of the ubiquitous Ca$\sp{++}$-binding protein calmodulin (CaM) with microtubules (MTs) in the mammalian mitotic apparatus (MA). Fluoresence conjugates of CaM with tetramethylrhodamine isothiocyanate (CaM-TRITC) and tubulin with dichlorotriazinyl aminofluorescein (tubulin-DTAF) were prepared and microinjected into living PtK$\sb1$ cells. A low-light video camera coupled to a fluorescence microscope and interfaced to a digital image processing system was used to record and compare the distribution of each of these proteins within mitotic cells. Serial sections of microinjected cells were made for transmission electron microscopy (TEM) in order to allow comparison of fluorescent images with cell ultrastructure. We confirmed that the distribution of fluorescent tubulin coincided with regions containing MTs in the TEM sections. We used exposure to cold or the MT inhibitor nocodazole to selectively eliminate or alter spindle MTs. After cold treatment, or treatment with a low concentration (0.03 $\mu$g/ml) of nocodazole, the only MTs which remained appeared to be kinetochore MTs (kMTs). The CaM distribution in these cells was identical to the localization seen in untreated cells, and appeared to be co-localized with the kMTs. When a higher concentration (3.0 $\mu$g/ml) of nocodazole was used, CaM co-localized with what appeared to be remnants of kMTs. We found that when either CaM, CaM-TRITC, or a Ca$\sp{++}$-insensitive fluorescent analog of CaM (HA-CaM-TRITC) were microinjected into mitotic cells prior to treatment with nocodazole, the kMTs became transiently resistant to the depolymerizing effect of the drug. In a permeabilized cell system, in the presence of 10 mM EGTA, both CaM-TRITC and HA-CaM-TRITC were observed to concentrate in an apparently normal distribution in mitotic cells. Finally, in cells recovering from nocodazole treatment sufficient to remove all of the cellular MTs, CaM-TRITC and tubulin-DTAF were observed to co-localize near centrosomes and kinetochores, in regions which contained newly polymerized MTs. Later in the recovery period, when normal appearing spindles had formed, CaM attained its usual distribution, concentrated primarily in pole-proximal regions. Based on these observations, we conclude that CaM achieves a structural association with kMTs in the MA in the absence of Ca$\sp{++}$ and suggest that the initial effect of this association is to increase the stability of kMTs. Moreover, the original hypothesis for the role of CaM in mitosis, which predicts that CaM acts in the MA to mediate a Ca$\sp{++}$-dependent MT depolymerization, needs revision in order to account for our observations.