The Drosophila CLASP homologue, Mast/Orbit regulates the dynamic behaviour of interphase microtubules by promoting the pause state

Abstract

An important group of microtubule associated proteins are the plus-end tracking proteins which includes the Mast/Orbit/CLASPs family amongst others. Several of these proteins have important functions during interphase and mitosis in the modulation of the dynamic properties of microtubules, however, the precise mechanism remains to be elucidated. To investigate the role of Mast in the regulation of microtubule behaviour during interphase, we used RNAi in Drosophila S2 culture cells stably expressing GFP-alpha-tubulin and followed the behaviour of microtubules in vivo. Mast depleted cells show a significant reduction of microtubule density and an abnormal interphase microtubule array that rarely reaches the cell cortex. Analysis of the dynamic parameters revealed that in the absence of Mast, microtubules are highly dynamic, constantly growing or shrinking. These alterations are characterized by a severe reduction in the transition frequencies to and from the pause state. Moreover, analysis of de novo microtubule polymerization after cold treatment showed that Mast is not required for nucleation since Mast depleted cells nucleate microtubules soon after return to normal temperature. Taken together these results suggest that Mast plays an essential role in reducing the dynamic behaviour of microtubules by specifically promoting the pause state.

Parallel Genetic and Proteomic Screens Identify Msps as a CLASP–Abl Pathway Interactor in Drosophila

“…Msps interacts with CLASP in both the genetic and proteomic screens, and it antagonizes CLASP and Abl signaling. The antagonism seen between Msps and CLASP in the Drosophila retina is consistent with cell culture studies, which have shown that CLASP regulates MT dynamics by specifically promoting the pause state (Sousa et al 2007) whereas Mspsfamily proteins function as MT antipause factors (Brittle and Ohkura 2005). More specifically, CLASPs have MT-stabilizing effects (Drabek et al 2006), and depleting cells of CLASP protein results in highly dynamic, constantly growing or shrinking MTs (Sousa et al 2007).…”

“…The antagonism seen between Msps and CLASP in the Drosophila retina is consistent with cell culture studies, which have shown that CLASP regulates MT dynamics by specifically promoting the pause state (Sousa et al 2007) whereas Mspsfamily proteins function as MT antipause factors (Brittle and Ohkura 2005). More specifically, CLASPs have MT-stabilizing effects (Drabek et al 2006), and depleting cells of CLASP protein results in highly dynamic, constantly growing or shrinking MTs (Sousa et al 2007). Alternatively, Msps family members can have the opposite effect (Popov and Karsenti 2003), catalyzing the addition and removal of multiple tubulin dimers at MT plus-ends (Brouhard et al 2008), and depletion of Msps results in a dramatic increase in MT pausing with little or no growth (Brittle and Ohkura 2005).…”

“…While their overlapping subcellular location suggests a possible association, Msps has not previously been shown to interact physically or functionally with CLASP in any system. Moreover, while CLASP-family proteins have been described as MT ''pause'' factors (Mimori-Kiyosue et al 2005;Sousa et al 2007) that reduce the dynamics of MT extension and retraction, Msps-family proteins have been called MT ''antipause'' factors (Brittle and Ohkura 2005), promoting rapid MT dynamics. These reciprocal functions in the regulation of MT dynamics in other systems suggest a possible antagonistic relationship between CLASP and Msps, consistent with the polarity of genetic interaction initially detected in our system.…”

Dynein and Mast/Orbit/CLASP have antagonistic roles in regulating kinetochore-microtubule plus-end dynamics

“…They belong to a subfamily of MAPs known as plus-end tracking proteins (+TIPs) (Carvalho et al, 2003) that bind microtubules and transiently accumulate at their growing plus-ends (Akhmanova et al, 2001;Sousa et al, 2007). They also localize to kinetochores of mitotic chromosomes, centrosomes, the central spindle region and the midbody (Lemos et al, 2000;Maiato et al, 2003a;Pereira et al, 2006).…”

“…They also localize to kinetochores of mitotic chromosomes, centrosomes, the central spindle region and the midbody (Lemos et al, 2000;Maiato et al, 2003a;Pereira et al, 2006). Recently, it was shown that in interphase Mast has a stabilizing role on microtubules by promoting the pause state (Sousa et al, 2007). In mitosis, Mast was shown to be required for functional kinetochore-microtubule attachments, chromosome congression and maintenance of spindle bipolarity (Lemos et al, 2000;Maiato et al, 2002).…”