HURP was recently identified as a Ran-target, both in human cells (Sillje et al., 2006
) and in Xenopus
egg extracts, where it is the essential component of a complex required for the bipolarization of Ran spindles. The HURP complex forms upon binding to MTs (Supplemental Figure S3A and Koffa et al., 2006
). However, immunoprecipitations performed either in HeLa cell extracts (Wong et al., 2008
) or in complete Xenopus
egg extracts in the absence of prepolymerized MTs suggest that HURP also exists in a free state. Consistent with this, in complete extracts, none of the components of the HURP complex are detected by Western blotting in HURP immunoprecipitations (Supplemental Figure S3B and our unpublished data). Sequencing of coimmunoprecipitated proteins by mass spectrometry also failed to identify any members of the HURP complex, whereas importin β, another known HURP interactor (Sillje et al., 2006
), was found (our unpublished data). Moreover, although the MAP fraction where HURP is quantitatively present in the complex is active in promoting bipolarization of Ran-induced asters (Koffa et al., 2006
), recombinant HURP is unable to do so (our unpublished data). We therefore sought to analyze whether free HURP also has a function by performing immunodepletion and restoration in Xenopus
egg extracts, an approach that revealed a novel HURP function during early meiotic spindle assembly.
We first addressed the role of HURP in spindle formation in the presence of both inducers of MT assembly, centrosomes, and chromatin. HURP depletion severely impaired bipolar spindle formation (, B and C). The specificity of these observations is proven by restoration of bipolarity on addition of xlHURP (, B and C). Interestingly, in the few bipolar structures that could still form upon HURP depletion, we observed a significant decrease in kinetochore and interpolar MT overlaps in the spindle midzone, whereas astral MTs did not seem to be affected (B, ΔHURP, right panel).
To elucidate the molecular mechanism of these HURP effects, we examined the role of HURP in either centrosome- or chromatin-dependent MT assembly. We tested whether and how HURP influences MT growth from centrosomes, in the light of HURP's reported function as an MT stabilizer (Koffa et al., 2006
; Sillje et al., 2006
; Wong and Fang, 2006
; Santarella et al., 2007
). In particular, we tested whether HURP behaves as a plus-end MT stabilizer. HURP depletion did not affect centrosomal aster length (), even in the absence of active TPX2 or the presence of excess RanGTP. These results indicate that HURP does not contribute to centrosome-dependent MT growth or plus-end stabilization, in contrast to XMAP215 or Cdk11 (Niethammer et al., 2007
). However, HURP-depleted extracts were almost completely unable to generate bipolar spindles around chromatin beads (, A and B). The chromatin-dependent pathway of spindle assembly operates through the production of RanGTP. Interestingly, neither Ran-asters nor Ran-spindles formed in HURP-depleted extracts (C). Therefore, HURP depletion results in a much stronger phenotype than that seen after HURP antibody inhibition (Koffa et al., 2006
). This argues that previous antibody addition did not completely inactivate HURP and that sufficient amounts of HURP remained free and active to allow the formation of partly organized structures. The fact that HURP has a very early role in chromatin-mediated MT assembly is supported by the fact that MCAK depletion does not rescue HURP depletion (B), which clearly shows that HURP activity in MT assembly precedes that of MCAK.
Taken together these results indicate that HURP works as a direct chromatin/RanGTP-target in early MT assembly, similarly to TPX2 (Wittmann et al., 2000
; Gruss et al., 2001
). We have previously shown that the two proteins belong to the Aurora A kinase-dependent HURP complex that forms on polymerized MTs (Koffa et al., 2006
) and that functions in the bipolarization of Xenopus
mitotic spindles. In the absence of MT, in total Xenopus
extracts, the two MAPs do not detectably interact with each other (Supplemental Figure S3). However, both HURP and TPX2 are very abundant proteins, and it was possible that minor fractions of the two proteins interact. However, even in fractions that were highly enriched for HURP and TPX2, we failed to detect any interaction between the proteins in the absence of MTs. These biochemical conclusions are in line with localization studies showing that HURP and TPX2 are enriched on distinct domains of the mitotic spindle: HURP remains at the spindle midzone, whereas TPX2 moves toward spindle poles (, A and B, mock panels). Furthermore, this localization is preserved upon depletion of the other MAP, suggesting that the two proteins work and localize independently of each other (, A and B, ΔHURP and ΔTPX2 panels). This reflects the fact that, even though both are required for early steps of meiotic spindle MT assembly, HURP and TPX2 carry out distinct later functions in association with different partners: TPX2 is known to be transported to the spindle poles by the dynein–dynactin complex (Wittmann et al., 2000
), whereas HURP has been shown to interact with the plus end motor protein Eg5 (Koffa et al., 2006
) that also localizes to the spindle midzone (Sawin and Mitchison, 1995
; Lockhart and Cross, 1996
; Blangy et al., 1997
). At later stages of spindle assembly TPX2 is required, together with Xklp2 and dynein, for MT focusing and spindle pole organization (Wittmann et al., 2000
), whereas HURP is crucial for spindle bipolarization, metaphase chromosome alignment, and stabilization of kinetochore fibers (Koffa et al., 2006
; Sillje et al., 2006
In conclusion, we show here that HURP fulfills an essential function in the early steps of MT assembly driven by chromatin/RanGTP similar to, but independent of TPX2.