CLASPs function redundantly to regulate astral microtubules in the C. elegans embryo

Abstract

Microtubule dynamics are thought to play an important role in regulating microtubule interactions with cortical force generating motor proteins that position the spindle during asymmetric cell division. CLASPs are microtubule-associated proteins that have a conserved role in regulating microtubule dynamics in diverse cell types. Caenorhabditis elegans has three CLASP homologs in its genome. CLS-2 is known to localize to kinetochores and is needed for chromosome segregation at meiosis and mitosis; however CLS-1 and CLS-3 have not been reported to have any role in embryonic development. Here, we show that depletion of CLS-2 in combination with either CLS-1 or CLS-3 results in defects in nuclear rotation, maintenance of spindle length, and spindle displacement in the one-cell embryo. Polarity is normal in these embryos, but reduced numbers of astral microtubules reach all regions of the cortex at the time of spindle positioning. Analysis of the microtubule plus-end tracker EB1 also revealed a reduced number of growing microtubules reaching the cortex in CLASP depleted embryos, but the polymerization rate of astral microtubules was not slower than in wild type. These results indicate that C. elegans CLASPs act partially redundantly to regulate astral microtubules and position the spindle during asymmetric cell division. Further, we show that these spindle pole-positioning roles are independent of the CLS-2 binding proteins HCP-1 and HCP-2.

RanGTP and CLASP1 cooperate to position the mitotic spindle

“…We then quantified the number of astral MTs observed to make contact with the cell cortex. In agreement with the literature (Mimori-Kiyosue et al , 2005; Espiritu et al , 2012), we found that overexpression of CLASP1 resulted in a significant increase in the number of astral MTs contacting the cortex (Figure 5B). In contrast, disruption of the Ran pathway with importazole caused a significant decrease in astral MT/cortex contacts.…”

“…CLASP1 was shown to be important for spindle positioning in multiple organisms, likely due to stabilization of end-on astral MT attachments with the cell cortex (Mimori-Kiyosue et al , 2005; Samora et al , 2011; Espiritu et al , 2012). Our work demonstrates that CLASP1 overexpression can rescue astral MT/cortex contacts in importazole-treated cells without affecting MT elongation speed, showing that RanGTP and CLASP1 affect astral MT dynamics through different mechanisms.…”

“…CLASP proteins regulate MT dynamics by promoting MT rescue and suppressing MT catastrophe through the activity of conserved TOG domains, which recruit tubulin dimers to MT plus ends (reviewed in Al-Bassam and Chang, 2011). Recent work showed that CLASP1 activity is required for proper positioning of the mitotic spindle in both the C. elegans embryo and mammalian cells, but the relationship between the CLASP1 and RanGTP regulated spindle-positioning pathways is unclear (Samora et al , 2011; Espiritu et al , 2012). …”

In Situ Imaging in C. elegans Reveals Developmental Regulation of Microtubule Dynamics

“…These targets included, as expected, proteins involved in centrosome duplication and thus cell division (three centrosomal proteins, SAS-3 SAS-4 and SAS-6, plus-end binding proteins CLS-1 CLASP and EBP-2 EB1 , and TPXL-1 TPX2 ; Table S2; (Espiritu et al, 2012; Kirkham et al, 2003; Leidel et al, 2005; Rogers et al, 2002; Wittmann et al, 2000). The milder effect resulting from depletion of the same proteins later in development suggests that these proteins are largely dispensable for egg-laying apparatus function.…”

How to be at the right place at the right time: The importance of spindle positioning in embryos

“…Laser‐severing experiments have additionally shown that the pulling forces in the posterior end of the cell are stronger than those found in the anterior, resulting in spindle displacement from a central location (Grill et al, ; reviewed in Gillies and Cabernard, ). Other studies demonstrated the importance of the CLASP proteins, microtubule‐associated proteins that are necessary for the astral microtubules to reach the cell cortex at the right time during spindle positioning (Espiritu et al, ). Thus, data from the C. elegans embryo overwhelmingly favors a role for pulling forces in generating the asymmetric spindle position during their first division.…”