Multifactorial characterization of organelle motility patterns reveals the role of peroxin proteins in actomyosin-dependent peroxisome movement in plants.

Plant subcellular architecture is dynamic, as organelles shift, morph, and move to accommodate the fluctuating needs of plant physiology.  Organelle motility and distribution are fundamental to the coordination of plant development, metabolism, and stress response. However, the complexity of organelle movement requires nuanced characterization beyond velocity to detect subtle but critical differences among cell types, developmental stages, and environmental conditions. While severe motility phenotypes—like complete stagnation or substantial reductions in velocity—enabled the discovery of Myosin XI motors as the primary drivers of organelle movement in plant cells, the complex mechanisms of organelle-specific movement and its regulation remain unclear. Using spinning disk confocal microscopy, I captured peroxisomes, mitochondria, and Golgi at 250 ms intervals, with markers co-expressed simultaneously for direct comparison among organelles. Thousands of organelles in both tobacco and Arabidopsis epidermis and mesophyll were tracked to assess motion patterns according to velocity, distance, area, and linearity. Peroxisomes display distinct motility patterns from Golgi and mitochondria in mesophyll and are less mobile in the mesophyll than epidermis in Arabidopsis, likely due to their predominant role in photorespiration that requires physical coordination among different organelle types in mesophyll cells. While Myosin XI proteins transport cargo indiscriminately, the distinct motility of peroxisomes indicates a regulatory mechanism for peroxisome-specific actomyosin movement. To this end, I identified several peroxisome-localized Myosin XI-K (XIK) interactors using BiFC and analyzed peroxisome movement in T-DNA mutants of the corresponding genes. My results reveal that Peroxin 14 (PEX14), a peroxisome biogenesis factor, may function as an adaptor for peroxisome-specific recruitment of myosin motors. Additional peroxin proteins, also identified as XIK interactors, likely contribute to peroxisome motility as well. Overall, this multifactorial approach for studying organelle motility unveils a novel function for some peroxisome protein import factors and opens vast possibilities for discovering the regulation of plant subcellular dynamics.