Podosome dynamics and location in vascular smooth muscle cells require CLASP-dependent microtubule bending.

Extracellular matrix (ECM) remodeling during physiological processes is mediated by invasive protrusions called podosomes. Positioning and dynamics of podosomes define the extent of ECM degradation. Microtubules are known to be involved in podosome regulation, but the role of microtubule (MT) network configuration in podosome dynamics and positioning is not well understood. Here, we show that the arrangement of the microtubule network defines the pattern of podosome formation and relocation in vascular smooth muscle cells (VSMCs). We show that microtubule plus-end targeting facilitates de novo formation of podosomes, in addition to podosome remodeling. Moreover, specialized bent microtubules with plus ends reversed towards the cell center promote relocation of podosomes from the cell edge to the cell center, resulting in an evenly distributed podosome pattern. Microtubule bending is induced downstream of protein kinase C (PKC) activation and requires microtubule-stabilizing proteins known as cytoplasmic linker associated proteins (CLASPs) and retrograde actin flow. Similar to microtubule depolymerization, CLASP depletion by siRNA blocks microtubule bending and eliminates centripetal relocation of podosomes. Podosome relocation also coincides with translocation of podosome-stimulating kinesin KIF1C, which is known to move preferentially along CLASP-associated microtubules. These findings indicate that CLASP-dependent microtubule network configuration is critical to the cellular location and distribution of KIF1C-dependent podosomes.