We utilized automated image analysis algorithms to screen for regulators of MT assembly dynamics induced downstream of Rac1 activation. Our results suggest that Op18/stathmin, the dynactin component p150glued
, the +TIP proteins APC2 and EB1, the MT severing enzyme Spastin, and the kinase MARK2 may be potential downstream mediators of Rac1 signaling that regulate MT dynamics. Mechanistic links between Rac1 and APC or Op18 have previously been reported 
, however the other factors identified here represent novel potential Rac1 targets. Thus, our results suggest that Rac1 acts as a master regulator of leading edge MT dynamics through multiple molecular pathways.
Our study represents the first time that parameters of MT growth dynamics have been used as the basis of a phenotypic screen, and was made possible by the development of automated image analysis programs 
. This method allowed measurement of MT growth excursions throughout the cell, including short-lived, slow growth excursions that were not measurable by hand-tracking in previous studies 
, suggesting that activated Rac1 primarily promotes less persistent and slower polymerization of MTs, especially in the leading edge ().
By further analyzing MT growth orientation in lamellipodia, we identified a single protein, MARK2, as required for Rac1 effects on both MT growth dynamics and orientation mediating pioneer MT formation. MARK2 is the mammalian homologue of the polarity protein PAR-1 which is thought to mediate polarity through effects on MTs 
. However, this is the first report of MARK2/PAR1’s effects on polarity and MT dynamics being controlled by the Rac1 signaling cascade. We found that RNAis targeting any of the three MARK isoforms (1, 2, and 3) promoted fast MT assembly, suggesting that MARKs function to slow MT growth or destabilize MTs in situ, in agreement with previous studies 
. Interestingly, depletion of MARK 1, 2 or 3 suppressed the slow, short-lived growth induced by CA-Rac1, but only depletion of MARK2 suppressed the effects of Rac1 on MT orientation in lamellipodia. Taken together, this suggests that all three MARK isoforms function downstream of Rac1 to regulate MT growth, but MARK2 additionally regulates Rac-1 mediated MT orientation.
The molecular mechanism by which Rac1 promotes MARK2-mediated regulation of MT growth dynamics and orientation in the leading edge is of interest. MARK2 is known to be activated by phosphorylation 
, and active MARK2 in turn phosphorylates MT-associated proteins (MAPs) such as tau, MAP2 and MAP4, which causes them to dissociate from MTs 
. Notably, we found that RNAis targeting several MAPs including MAP4, MAP1B and MAP2, reduced the proportion of slow, short-lived MT growth excursions (), similar to the effects of MARK2 depletion. Further study is needed to determine if Rac1 mediates local downstream inactivation of MARK2 in lamellipodia to reduce MAP phosphorylation and promote MAP-MT binding to locally stabilize MTs and drive pioneer MT growth. In addition, although we showed that Rac1 activity controls MARK2 localization and Rac1’s effects on MTs require MARK2, it would be of interest to additionally know if Rac1 controls MARK2’s kinase activity. MARK2 has been shown to interact with the Rac1 targets, GSK3β and a PAK kinase 
, and these in turn regulate Op18/stathmin and CLASP 
. This suggests that the MARK2, GSK3β, and PAK pathways of MT regulation could intersect downstream of Rac1.
The question of how MARK2 regulates MT orientation in lamellipodia is unclear. Since MT bending and growth parallel to the cell edge is thought to be driven by MT interactions with lamellipodial F-actin retrograde flow 
, it is possible that MARK2’s effect on pioneer MT orientation are mediated by phosphoregulation of MAPs that bind both MTs and actin, as has been demonstrated for ACF7, APC and MAP2 
. Alternatively, MARK2 may coordinately regulate MT and actin cytoskeletal dynamics through Pak kinases, which target both MT and actin binding proteins 
Our study builds on previous work showing that MARK kinases and their PAR1 homologues regulate cell polarity through downstream effects on MTs 
. In a recent study of dendritic spines, it was found that that knockdown of the MARK2 homologue PAR-1 decreased the distance and duration of MT growth, as analyzed by tracking fluorescent +TIP proteins 
. This contrasts our study in which MT growth persistence was increased in MARK2 knock-down cells (, and ). This suggests that MARK2 may utilize distinct effectors to control MT dynamics differently in each system. Notably, tracking of GFP-EB3 dynamics in Drosophila
wing epidermis 
and oocytes 
revealed that the MARK2 homologue, Par-1, plays a crucial role in establishing a small bias in the orientation of growing MTs without a substantial change in overall MT distribution, resulting in asymmetric segregation of polarity proteins. In addition, similar to lamellipodia in migrating cells, MTs tend to bend and grow along the posterior cortex of Drosophila
oocytes where Par-1 localizes, and Par-1 mutants fail to show these polarized MT growth excursions 
. These data indicate that MARK2/PAR-1 has a conserved role in organizing MTs and regulating their growth and orientation in many systems.
Consistent with the role of MARK2 in MT reorganization, our results demonstrate that MARK2 is essential for cell polarization and directed cell migration. We found that MARK2-depleted cells in a wound edge have reduced pioneer MTs in the leading lamellipodia and show faster MT growth (). Indeed, depletion of MARK2 caused a failure in centrosome polarization towards the wound edge, and inhibited migration velocity and directionality (). MARK2/PAR-1 has also been shown to play a crucial role in directed migration in other cell types. For example, in the cerebral cortex, neurons depleted of MARK2 exhibit altered directed migration and reduced centrosome motility 
. In addition, par-1
null mutants in Drosophila
border cells also fail in directed cell migration, showing abnormal protrusion in their front 
. Therefore, MARK2/PAR-1 is a conserved key mediator for establishing cell polarity during directed migration, likely through regulating MT dynamics under the control of Rac1.
Taken together, our study shows the power of high-resolution, quantitative live cell imaging assays for refined screening of protein function, and identifies MARK2 as essential for linking Rac1 activation to polarization of MTs and their assembly dynamics critical to directed cell migration.