No abstract

Microtubules of all eukaryotic cells are formed by α- and β-tubulin heterodimers. In addition to the well known cytoplasmic tubulins, a subpopulation of tubulin can occur in the nucleus. So far, the potential function of nuclear tubulin has remained elusive. In this work, we show that α- and β-tubulins of various organisms contain multiple conserved nuclear export sequences, which are potential targets of the Exportin 1/CRM1 pathway. We demonstrate exemplarily that these NES motifs are sufficient to mediate export of GFP as model cargo and that this export can be inhibited by leptomycin B, an inhibitor of the Exportin 1/CRM1 pathway. Likewise, leptomycin B causes accumulation of GFP-tagged tubulin in interphase nuclei, in both plant and animal model cells. Our analysis of nuclear tubulin content supports the hypothesis that an important function of nuclear tubulin export is the exclusion of tubulin from interphase nuclei, after being trapped by nuclear envelope reassembly during telophase.

The ARP2/3 complex and formins are the only known plant actin nucleators. Besides their actin-related functions, both systems also modulate microtubule organization and dynamics. Loss of the main housekeeping Arabidopsis thaliana Class I membranetargeted formin FH1 (At3g25500) is known to increase cotyledon pavement cell lobing, while mutations affecting ARP2/3 subunits exhibit an opposite effect. Here we examine the role of FH1 and the ARP2/3 complex subunit ARPC5 (At4g01710) in epidermal cell morphogenesis with focus on pavement cells and trichomes using a model system of single fh1 and arpc5, as well as double fh1 arpc5 mutants. While cotyledon pavement cell shape in double mutants mostly resembled single arpc5 mutants, analysis of true leaf epidermal morphology, as well as actin and microtubule organization and dynamics, revealed a more complex relationship between the two systems and similar, rather than antagonistic, effects on some parameters. Both fh1 and arpc5 mutations increased actin network density and increased cell shape complexity in pavement cells and trichomes of first true leaves, in contrast to cotyledons. Thus, while the two actin nucleation systems have complementary roles in some aspects of cell morphogenesis in cotyledon pavement cells, they may act in parallel in other cell types and developmental stages.

ARP2/3 is a heteroheptameric protein complex evolutionary conserved in all eukaryotic organisms. Its conserved role is based on the induction of actin polymerization at the interface between membranes and the cytoplasm. Plant ARP2/3 has been reported to participate in actin reorganization at the plasma membrane during polarized growth of trichomes and at the plasma membrane-endoplasmic reticulum contact sites. We demonstrate here that individual plant subunits of ARP2/3 fused to fluorescent proteins form motile dot-like structures in the cytoplasm that are associated with plant peroxisomes. ARP2/3 dot structure is found at the peroxisome periphery and contains assembled ARP2/3 complex and WAVE/SCAR complex subunit NAP1. This dot occasionally colocalizes with the autophagosome, and under conditions that affect the autophagy, colocalization between ARP2/3 and the autophagosome increases. ARP2/3 subunits co-immunoprecipitate with ATG8f marker. Since mutants lacking functional ARP2/3 complex have more peroxisomes than WT, we link the ARP2/3 complex on peroxisomes to the process of peroxisome degradation by autophagy called pexophagy. Additionally, several other peroxisomal proteins colocalize with ARP2/3 dot on plant peroxisomes. Our results suggest a specific role of ARP2/3 and actin in the peroxisome periphery, presumably in membrane remodelling. We hypothesize that this role of ARP2/3 aids processes at the peroxisome periphery such as peroxisome degradation through autophagy or regulation of peroxisomal proteins localization or function.

Protein complex Arp2/3 has a conserved role in the nucleation of branched actin filaments. It is constituted of seven subunits, including actin-like subunits ARP2 and ARP3 plus five other subunits called Arp2/3 Complex Component 1 to 5, which are not related to actin. Knock-out plant mutants lacking individual plant ARP2/3 subunits have a typical phenotype of distorted trichomes, altered pavement cells shape and defects in cell adhesion. While knock-out mutant Arabidopsis plants for most ARP2/3 subunits have been characterized before, Arabidopsis plant mutants missing ARPC1 and ARPC3 subunits have not yet been described. Using CRISPR/Cas9, we generated knock-out mutants lacking ARPC1 and ARPC3 subunits. We confirmed that the loss of ARPC1 subunits results in the typical ARP2/3 mutant phenotype. However, the mutants lacking ARPC3 subunits resulted in plants with surprisingly different phenotypes. Our results suggest that plant ARP2/3 complex function in trichome shaping does not require ARPC3 subunit, while the fully assembled complex is necessary for the establishment of correct cell adhesion in the epidermis.