Jelinkova, B.; Voloshina, M.; Liebezeit, K.; Krtkova, J.; Garcia-Gonzalez, J.; Vosolsobe, S.; Harmanec, A.; Kollarova, E.; Baquero Forero, A.; Petrasek, J.; Schwarzerova, K.

The ARP2/3 complex generates branched actin networks that regulate membrane dynamics across eukaryotes. In plants, ARP2/3 is activated primarily by the WAVE/SCAR complex and is essential for cell morphogenesis, yet its spatiotemporal behavior in living cells remains poorly understood. Using high-resolution microscopy, we show that, in addition to the previously reported stable accumulation of WAVE/SCAR at three-way cell junctions and of WAVE/SCAR and ARP2/3 at peroxisomes, the subunits of both complexes are also present in the cortical cytoplasm in the form of dynamic, and short-lived assemblies with an average lifetime in order of seconds. Genetic and colocalization analyses demonstrated that only a minority of observed complexes are fully assembled and active, indicating the presence of a large pool of partially assembled or inactive structures. Cytoskeletal inhibitors revealed that microtubules influence foci density, whereas actin primarily affects their dynamics, suggesting coordinated regulation between cytoskeletal systems. Importantly, our analysis demonstrated a spatial and functional association between dynamic ARP2/3 foci and exocytotic events at the plasma membrane.