skip to main content

Title: O -glycosylation of the extracellular domain of pollen class I formins modulates their plasma membrane mobility

In plant cells, linkage between the cytoskeleton, plasma membrane, and cell wall is crucial for maintaining cell shape. In highly polarized pollen tubes, this coordination is especially important to allow rapid tip growth and successful fertilization. Class I formins contain cytoplasmic actin-nucleating formin homology domains as well as a proline-rich extracellular domain and are candidate coordination factors. Here, using Arabidopsis, we investigated the functional significance of the extracellular domain of two pollen-expressed class I formins: AtFH3, which does not have a polar localization, and AtFH5, which is limited to the growing tip region. We show that the extracellular domain of both is necessary for their function, and identify distinct O-glycans attached to these sequences, AtFH5 being hydroxyproline-arabinosylated and AtFH3 carrying arabinogalactan chains. Loss of hydroxyproline arabinosylation altered the plasma membrane localization of AtFH5 and disrupted actin cytoskeleton organization. Moreover, we show that O-glycans differentially affect lateral mobility in the plasma membrane. Together, our results support a model of protein sub-functionalization in which AtFH5 and AtFH3, restricted to specific plasma membrane domains by their extracellular domains and the glycans attached to them, organize distinct subarrays of actin during pollen tube elongation.

more » « less
Award ID(s):
Author(s) / Creator(s):
; ; ;
Publisher / Repository:
Oxford University Press
Date Published:
Journal Name:
Journal of Experimental Botany
Page Range / eLocation ID:
p. 3929-3945
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. Abstract

    Root hairs are single-cell protrusions that enable roots to optimize nutrient and water acquisition. These structures attain their tubular shapes by confining growth to the cell apex, a process called tip growth. The actin cytoskeleton and endomembrane systems are essential for tip growth; however, little is known about how these cellular components coordinate their activities during this process. Here, we show that SPIRRIG (SPI), a beige and Chediak Higashi domain-containing protein involved in membrane trafficking, and BRK1 and SCAR2, subunits of the WAVE/SCAR (W/SC) actin nucleating promoting complex, display polarized localizations in Arabidopsis thaliana root hairs during distinct developmental stages. SPI accumulates at the root hair apex via post-Golgi compartments and positively regulates tip growth by maintaining tip-focused vesicle secretion and filamentous-actin integrity. BRK1 and SCAR2 on the other hand, mark the root hair initiation domain to specify the position of root hair emergence. Consistent with the localization data, tip growth was reduced in spi and the position of root hair emergence was disrupted in brk1 and scar1234. BRK1 depletion coincided with SPI accumulation as root hairs transitioned from initiation to tip growth. Taken together, our work uncovers a role for SPI in facilitating actin-dependent root hair development in Arabidopsis through pathways that might intersect with W/SC.

    more » « less
  2. Summary

    HYDROXYPROLINEO‐ARABINOSYLTRANSFERASEs (HPATs) initiate a post‐translational protein modification (Hyp‐Ara) found abundantly on cell wall structural proteins. InArabidopsis thaliana,HPAT1andHPAT3are redundantly required for full pollen fertility. In addition to the lack of Hyp‐Ara inhpat1/3pollen tubes (PTs), we also found broadly disrupted cell wall polymer distributions, particularly the conversion of the tip cell wall to a more shaft‐like state. Mutant PTs were slow growing and prone to rupture and morphological irregularities. In a forward mutagenesis screen for suppressors of thehpat1/3low seed‐set phenotype, we identified a missense mutation inexo70a2, a predicted member of the vesicle‐tethering exocyst complex. The suppressed pollen had increased fertility, fewer morphological defects and partially rescued cell wall organization. A transcriptional null allele ofexo70a2also suppressed thehpat1/3fertility phenotype, as did mutants of core exocyst complex membersec15a, indicating that reduced exocyst function bypassed the PT requirement for Hyp‐Ara. In a wild‐type background,exo70a2reduced male transmission efficiency, lowered pollen germination frequency and slowed PT elongation. EXO70A2 also localized to the PT tip plasma membrane, consistent with a role in exocyst‐mediated secretion. To monitor the trafficking of Hyp‐Ara modified proteins, we generated an HPAT‐targeted fluorescent secretion reporter. Reporter secretion was partially dependent onEXO70A2and was significantly increased inhpat1/3PTs compared with the wild type, but was reduced in the suppressedexo70a2 hpat1/3tubes.

    more » « less
  3. Coordination between actin and microtubules is important for numerous cellular processes in diverse eukaryotes. In plants, tip-growing cells require actin for cell expansion and microtubules for orientation of cell expansion, but how the two cytoskeletons are linked is an open question. In tip-growing cells of the moss Physcomitrella patens , we show that an actin cluster near the cell apex dictates the direction of rapid cell expansion. Formation of this structure depends on the convergence of microtubules near the cell tip. We discovered that microtubule convergence requires class VIII myosin function, and actin is necessary for myosin VIII–mediated focusing of microtubules. The loss of myosin VIII function affects both networks, indicating functional connections among the three cytoskeletal components. Our data suggest that microtubules direct localization of formins, actin nucleation factors, that generate actin filaments further focusing microtubules, thereby establishing a positive feedback loop ensuring that actin polymerization and cell expansion occur at a defined site resulting in persistent polarized growth. 
    more » « less
  4. Abstract The phragmoplast separates daughter cells during cytokinesis by constructing the cell plate, which depends on interaction between cytoskeleton and membrane compartments. Proteins responsible for these interactions remain unknown, but formins can link cytoskeleton with membranes and several members of formin protein family localize to the cell plate. Progress in functional characterization of formins in cytokinesis is hindered by functional redundancies within the large formin gene family. We addressed this limitation by employing Small Molecular Inhibitor of Formin Homology 2 (SMIFH2), a small-molecule inhibitor of formins. Treatment of tobacco (Nicotiana tabacum) tissue culture cells with SMIFH2 perturbed localization of actin at the cell plate; slowed down both microtubule polymerization and phragmoplast expansion; diminished association of dynamin-related proteins with the cell plate independently of actin and microtubules; and caused cell plate swelling. Another impact of SMIFH2 was shortening of the END BINDING1b (EB1b) and EB1c comets on the growing microtubule plus ends in N. tabacum tissue culture cells and Arabidopsis thaliana cotyledon epidermis cells. The shape of the EB1 comets in the SMIFH2-treated cells resembled that of the knockdown mutant of plant Xenopus Microtubule-Associated protein of 215 kDa (XMAP215) homolog MICROTUBULE ORGANIZATION 1/GEMINI 1 (MOR1/GEM1). This outcome suggests that formins promote elongation of tubulin flares on the growing plus ends. Formins AtFH1 (A. thaliana Formin Homology 1) and AtFH8 can also interact with EB1. Besides cytokinesis, formins function in the mitotic spindle assembly and metaphase to anaphase transition. Our data suggest that during cytokinesis formins function in: (1) promoting microtubule polymerization; (2) nucleating F-actin at the cell plate; (3) retaining dynamin-related proteins at the cell plate; and (4) remodeling of the cell plate membrane. 
    more » « less
  5. Plakophilin-3 is a ubiquitously expressed protein found widely in epithelial cells and is a critical component of desmosomes. The plakophilin-3 carboxy-terminal domain harbors nine armadillo repeat motifs with largely unknown functions. Here, we report the 5 Å cryogenic electron microscopy (cryoEM) structure of the armadillo repeat motif domain of plakophilin-3, one of the smaller cryoEM structures reported to date. We find that this domain is a monomer or homodimer in solution. In addition, using an in vitro actin co-sedimentation assay, we show that the armadillo repeat domain of plakophilin-3 directly interacts with F-actin. This feature, through direct interactions with actin filaments, could be responsible for the observed association of extra-desmosomal plakophilin-3 with the actin cytoskeleton directly attached to the adherens junctions in A431 epithelial cells. Further, we demonstrate, through lipid binding analyses, that plakophilin-3 can effectively be recruited to the plasma membrane through phosphatidylinositol-4,5-bisphosphate-mediated interactions. Collectively, we report on novel properties of plakophilin-3, which may be conserved throughout the plakophilin protein family and may be behind the roles of these proteins in cell–cell adhesion. 
    more » « less