In Chapter 5, we explored the ability of CLASP to organize dynamic actin filaments along microtubules.
We demonstrated that CLASP2 can template dynamic actin, bridging between microtubules (Figure 6.3).
Other crosslinking proteins of interest have been reported to facilitate similar crosslinking interactions, such
as septins. SEPT2/6/7 was very recently reported to be able to crosslink actin filaments to the microtubule latticein vitro(Nakos et al., 2022). Strikingly, SEPT2/6/7 are also able to recruit polymerizing actin fila- ments, as described here for CLASP2 (Chapter 5). In contrast to our observations, they also reported partial attachments of dynamic actin filaments where the unbound segment branches off at an angle (Nakos et al., 2022). However similar, there may be subtle differences between the crosslinking interactions facilitated by CLASPs and septins that remain to be explained. Septins are filamentous proteins that interact with multiple components of the cytoskeleton. Septins form a heteromeric complex suggesting multivalent interactions with microtubules and actin filaments (Nakos et al., 2022), allowing for crosslinking interactions. Our results for CLASP and that reported for septins, demonstrate that the ability for actin filaments to polymerize on microtubules is indicative for low-affinity, transient interactions with actin, as thought for decades (Griffith and Pollard, 1982). We measured relatively low affinities for the F-actin: CLASP interaction (Chapter 3), supporting the mechanism by which CLASPs are also able to facilitate actin polymerization templated by microtubules.
Figure 6.3: Cartoon representing dynamic organization of actin filaments as templated by microtubules Here, several instances of actin filament and microtubule co-organization are illustrated, i.e., actin filaments
growing along microtubules, continued actin filament elongation from microtubule ends, and linking microtubules together by actin filaments bridges. These interactions were observed in this work and reported
for other proteins involved in microtubule-actin crosstalk.
Another interesting potential role of CLASPs in these interactions include mechanisms from a recent report establishing how the +TIP complex, i.e. CLIP-170, EB, and mDia1, helps govern actin filament orga- nization by microtubules (Henty-Ridilla et al., 2016). Here, the authors demonstrate how CLIP-170 localizes to growing microtubule ends by EB and bind mDia1 to initiate actin polymerization from microtubules.
CLIP-170 was suggested to interact with mDia1 through the formin elongation effector domain (FEED)
sequence within the protein. These interactions are of particular interest due to the connections between CLASPs and CLIPs. CLASPs were first discovered by identifying the binding partners of CLIP-170 and CLIP-115, where CLASPs were shown to directly interact with CLIPs and microtubules (Akhmanova et al., 2001). Like CLASPs, CLIP-170 regulates the microtubule network by tip-tracking microtubulesin vitroand in cells (Bieling et al., 2008; Dixit et al., 2009; Folker et al., 2005). Remarkably, CLIP-170 has also been reported to directly interact with actin, though appears to be unable to simultaneously bind microtubules and actin filaments as established with CLASPs in our study (Wu et al., 2021). This, however, does not rule out the possibility that in the cell, CLASPs and CLIPs work together to facilitate microtubule-actin crosstalk, such as described by Henty-Ridilla and colleagues. Potentially, CLASPs can help localize actin filaments to microtubules and interact with CLIPs at the end of the microtubule, where CLIPs attract formins through the FEED sequence to accelerate actin polymerization. In that context, CLASPs could further stabilize actin filament connections to microtubules through their crosslinking function. EBs provide additional anchoring of CLASPs and CLIPs to the microtubule end. The interplay between CLASPs, CLIPs, and EBs in these crosstalk interactions require further investigation.
Actin filaments connected to the microtubule ends have also been shown for APC, +TIPs, and branched actin networks as visualized using platinum replica electron microscopy (PREM) in hippocampal neurons (Efimova et al., 2020). Here, branched actin appeared to be connected to microtubule ends by immunogold labeled APC proteins in the growth cone. An additional density was identified to be a +TIP complex, which could include CLASPs. Furthermore, APC-mediated actin polymerization has been linked to focal adhe- sion dynamics. Here, human osteosarcoma cells with a separation of function APC mutant, abolishing actin nucleation, resulted in prolonged focal adhesion disassembly resulting from altered microtubule capture at focal adhesions and autophagosome transport (Juanes et al., 2017, 2019). CLASPs are also implicated in the disassembly of focal adhesions reported to facilitate interactions with the cell cortex (Efimova et al., 2014;
Lawrence et al., 2020; Stehbens et al., 2014). The crosstalk between focal adhesions, actin stress fibers, and microtubules is very important in cell motility.
Overall, our results suggest a role of CLASP2 in organizing actin filaments along microtubules. We propose a model where CLASP2 can facilitate crosslinking between microtubules and actin filaments, when concentrated along the microtubule lattice due to the strong affinity of CLASPs for microtubules. Concen- trated CLASP2 on microtubules provides a local abundance of weak F-actin binding sites, allowing for robust microtubule and actin coalignment. We determined that the minimal CLASP construct has a relatively low affinity for F-actin, which supports the ability of concentrated CLASP2 to crosslink actin filaments, as well
as support actin filament elongation along microtubules.