Alexander R. Paredez

Visualization of Cellulose Synthase Demonstrates Functional Association with Microtubules

Expression of a functional yellow fluorescent protein fusion to cellulose synthase (CESA) in transgenic Arabidopsis plants allowed the process of cellulose deposition to be visualized in living cells. Spinning disk confocal microscopy revealed that CESA complexes in the plasma membrane moved at constant rates in linear tracks that were aligned and were coincident with cortical microtubules. Within each observed linear track, complex movement was bidirectional. Inhibition of microtubule polymerization changed the fine-scale distribution and pattern of moving CESA complexes in the membrane, indicating a relatively direct mechanism for guidance of cellulose deposition by the cytoskeleton.

Arabidopsis cortical microtubules position cellulose synthase delivery to the plasma membrane and interact with cellulose synthase trafficking compartments.

Plant cell morphogenesis relies on the organization and function of two polymer arrays separated by the plasma membrane: the cortical microtubule cytoskeleton and cellulose microfibrils in the cell wall. Studies using in vivo markers confirmed that one function of the cortical microtubule array is to drive organization of cellulose microfibrils by guiding the trajectories of active cellulose synthase (CESA) complexes in the plasma membrane, thus orienting nascent microfibrils. Here we provide evidence that cortical microtubules also position the delivery of CESA complexes to the plasma membrane and interact with small CESA-containing compartments by a mechanism that permits motility driven by microtubule depolymerization. The association of CESA compartments with cortical microtubules was greatly enhanced during osmotic stress and other treatments that limit cellulose synthesis. On recovery from osmotic stress, delivery of CESA complexes to the plasma membrane was observed in association with microtubule-tethered compartments. These results reveal multiple functions for the microtubule cortical array in organizing CESA in the cell cortex.

Genetic evidence for three unique components in primary cell-wall cellulose synthase complexes in Arabidopsis

In higher plants, cellulose is synthesized at the plasma membrane by the cellulose synthase (CESA) complex. The catalytic core of the complex is believed to be composed of three types of CESA subunits. Indirect evidence suggests that the complex associated with primary wall cellulose deposition consists of CESA1, -3, and -6 in Arabidopsis thaliana. However, phenotypes associated with mutations in two of these genes, CESA1 and -6, suggest unequal contribution by the different CESAs to overall enzymatic activity of the complex. We present evidence that the primary complex requires three unique types of components, CESA1-, CESA3-, and CESA6-related, for activity. Removal of any of these components results in gametophytic lethality due to pollen defects, demonstrating that primary-wall cellulose synthesis is necessary for pollen development. We also show that the CESA6-related CESAs are partially functionally redundant.

The Genome of Naegleria gruberi Illuminates Early Eukaryotic Versatility

Genome sequences of diverse free-living protists are essential for understanding eukaryotic evolution and molecular and cell biology. The free-living amoeboflagellate Naegleria gruberi belongs to a varied and ubiquitous protist clade (Heterolobosea) that diverged from other eukaryotic lineages over a billion years ago. Analysis of the 15,727 protein-coding genes encoded by Naeglerias 41 Mb nuclear genome indicates a capacity for both aerobic respiration and anaerobic metabolism with concomitant hydrogen production, with fundamental implications for the evolution of organelle metabolism. The Naegleria genome facilitates substantially broader phylogenomic comparisons of free-living eukaryotes than previously possible, allowing us to identify thousands of genes likely present in the pan-eukaryotic ancestor, with 40% likely eukaryotic inventions. Moreover, we construct a comprehensive catalog of amoeboid-motility genes. The Naegleria genome, analyzed in the context of other protists, reveals a remarkably complex ancestral eukaryote with a rich repertoire of cytoskeletal, sexual, signaling, and metabolic modules.

Genetic Evidence That Cellulose Synthase Activity Influences Microtubule Cortical Array Organization

To identify factors that influence cytoskeletal organization we screened for Arabidopsis (Arabidopsis thaliana) mutants that show hypersensitivity to the microtubule destabilizing drug oryzalin. We cloned the genes corresponding to two of the 131 mutant lines obtained. The genes encoded mutant alleles of PROCUSTE1 and KORRIGAN, which both encode proteins that have previously been implicated in cellulose synthesis. Analysis of microtubules in the mutants revealed that both mutants have altered orientation of root cortical microtubules. Similarly, isoxaben, an inhibitor of cellulose synthesis, also altered the orientation of cortical microtubules while exogenous cellulose degradation did not. Thus, our results substantiate that proteins involved in cell wall biosynthesis influence cytoskeletal organization and indicate that this influence on cortical microtubule stability and orientation is correlated with cellulose synthesis rather than the integrity of the cell wall.

An actin cytoskeleton with evolutionarily conserved functions in the absence of canonical actin-binding proteins

Giardia intestinalis, a human intestinal parasite and member of what is perhaps the earliest-diverging eukaryotic lineage, contains the most divergent eukaryotic actin identified to date and is the first eukaryote known to lack all canonical actin-binding proteins (ABPs). We sought to investigate the properties and functions of the actin cytoskeleton in Giardia to determine whether Giardia actin (giActin) has reduced or conserved roles in core cellular processes. In vitro polymerization of giActin produced filaments, indicating that this divergent actin is a true filament-forming actin. We generated an anti-giActin antibody to localize giActin throughout the cell cycle. GiActin localized to the cortex, nuclei, internal axonemes, and formed C-shaped filaments along the anterior of the cell and a flagella-bundling helix. These structures were regulated with the cell cycle and in encysting cells giActin was recruited to the Golgi-like cyst wall processing vesicles. Knockdown of giActin demonstrated that giActin functions in cell morphogenesis, membrane trafficking, and cytokinesis. Additionally, Giardia contains a single G protein, giRac, which affects the Giardia actin cytoskeleton independently of known target ABPs. These results imply that there exist ancestral and perhaps conserved roles for actin in core cellular processes that are independent of canonical ABPs. Of medical significance, the divergent giActin cytoskeleton is essential and commonly used actin-disrupting drugs do not depolymerize giActin structures. Therefore, the giActin cytoskeleton is a promising drug target for treating giardiasis, as we predict drugs that interfere with the Giardia actin cytoskeleton will not affect the mammalian host.

Prefoldin 6 is required for normal microtubule dynamics and organization in Arabidopsis

Newly translated tubulin molecules undergo a series of complex interactions with nascent chain-binding chaperones, including prefoldin (PFD) and chaperonin-containing TCP-1 (CCT). By screening for oryzalin hypersensitivity, we identified several mutants of Arabidopsis that have lesions in PFD subunits. The pfd6–1 mutant exhibits a range of microtubule defects, including hypersensitivity to oryzalin, defects in cell division, cortical array organization, and microtubule dynamicity. Consistent with phenotypic analysis, proteomic analysis indicates several isoforms of tubulins were reduced in pfd6–1. These results support the concept that the function of microtubules is critically dependent on the absolute amount of tubulins.

Identification of Obscure yet Conserved Actin-Associated Proteins in Giardia lamblia

ABSTRACT Consistent with its proposed status as an early branching eukaryote, Giardia has the most divergent actin of any eukaryote and lacks core actin regulators. Although conserved actin-binding proteins are missing from Giardia, its actin is utilized similarly to that of other eukaryotes and functions in core cellular processes such as cellular organization, endocytosis, and cytokinesis. We set out to identify actin-binding proteins in Giardia using affinity purification coupled with mass spectroscopy (multidimensional protein identification technology [MudPIT]) and have identified >80 putative actin-binding proteins. Several of these have homology to conserved proteins known to complex with actin for functions in the nucleus and flagella. We validated localization and interaction for seven of these proteins, including 14-3-3, a known cytoskeletal regulator with a controversial relationship to actin. Our results indicate that although Giardia lacks canonical actin-binding proteins, there is a conserved set of actin-interacting proteins that are evolutionarily indispensable and perhaps represent some of the earliest functions of the actin cytoskeleton.

Myosin-independent cytokinesis in Giardia utilizes flagella to coordinate force generation and direct membrane trafficking

Significance Many protists, including Giardia, lack myosin II and thus are unlikely to use the canonical contractile mechanism of cell division. Giardia depends solely on its flagella for motility; here, we show that flagella function is also required to drive daughter cells in opposite directions for cytokinesis. Additionally, just before cytokinesis, Rab11 accumulated in the forming furrow and the nascent intracytoplasmic axonemes were oriented to deliver Rab11. This mechanism constitutes a means to mark the center of the cell and guide trafficking to the furrow. These results support an emerging view that flagella play a central role in cell division among protists that lack myosin II. Devoid of all known canonical actin-binding proteins, the prevalent parasite Giardia lamblia uses an alternative mechanism for cytokinesis. Unique aspects of this mechanism can potentially be leveraged for therapeutic development. Here, live-cell imaging methods were developed for Giardia to establish division kinetics and the core division machinery. Surprisingly, Giardia cytokinesis occurred with a median time that is ∼60 times faster than mammalian cells. In contrast to cells that use a contractile ring, actin was not concentrated in the furrow and was not directly required for furrow progression. Live-cell imaging and morpholino depletion of axonemal Paralyzed Flagella 16 indicated that flagella-based forces initiated daughter cell separation and provided a source for membrane tension. Inhibition of membrane partitioning blocked furrow progression, indicating a requirement for membrane trafficking to support furrow advancement. Rab11 was found to load onto the intracytoplasmic axonemes late in mitosis and to accumulate near the ends of nascent axonemes. These developing axonemes were positioned to coordinate trafficking into the furrow and mark the center of the cell in lieu of a midbody/phragmoplast. We show that flagella motility, Rab11, and actin coordination are necessary for proper abscission. Organisms representing three of the five eukaryotic supergroups lack myosin II of the actomyosin contractile ring. These results support an emerging view that flagella play a central role in cell division among protists that lack myosin II and additionally implicate the broad use of membrane tension as a mechanism to drive abscission.

Rac Regulates Giardia lamblia Encystation by Coordinating Cyst Wall Protein Trafficking and Secretion

ABSTRACT Encystation of the common intestinal parasite Giardia lamblia involves the production, trafficking, and secretion of cyst wall material (CWM). However, the molecular mechanism responsible for the regulation of these sequential processes remains elusive. Here, we examined the role of GlRac, Giardia’s sole Rho family GTPase, in the regulation of endomembrane organization and cyst wall protein (CWP) trafficking. Localization studies indicated that GlRac is associated with the endoplasmic reticulum (ER) and the Golgi apparatus-like encystation-specific vesicles (ESVs). Constitutive GlRac signaling increased levels of the ER marker PDI2, induced ER swelling, reduced overall CWP1 production, and promoted the early maturation of ESVs. Quantitative analysis of cells expressing constitutively active hemagglutinin (HA)-tagged GlRac (HA-RacCA) revealed fewer but larger ESVs than control cells. Consistent with the phenotype of premature maturation of ESVs in HA-RacCA-expressing cells, constitutive GlRac signaling resulted in increased CWP1 secretion and, conversely, morpholino depletion of GlRac blocked CWP1 secretion. Wild-type cells unexpectedly secreted large quantities of CWP1 into the medium, and free CWP1 was used cooperatively during cyst formation. These results, in part, could account for the previously reported observation that G. lamblia encysts more efficiently at high cell densities. These studies of GlRac show that it regulates encystation at several levels, and our findings support its coordinating role as a regulator of CWP trafficking and secretion. The central role of GlRac in regulating membrane trafficking and the cytoskeleton, both of which are essential to Giardia parasitism, further suggests its potential as a novel target for drug development to treat giardiasis. IMPORTANCE The encystation process is crucial for the transmission of giardiasis and the life cycle of many protists. Encystation for Giardia lamblia involves the assembly of a protective cyst wall via sequential production, trafficking, and secretion of cyst wall material. However, the regulatory pathways that coordinate cargo maturation and secretion remain unknown. Here, we asked whether the signaling activities of G. lamblia’s single Rho family GTPase, GlRac, might have a regulatory role in the encystation process. We show that GlRac localizes to endomembranes and its signaling activities regulate the production of cyst wall protein 1 (CWP1), the maturation of encystation-specific vesicles (ESVs), and secretion of CWP1. We also show that secreted CWP1 is available for the development of cysts at the population level, a finding that in part could explain why Giardia encystation proceeds more efficiently at high cell densities. The encystation process is crucial for the transmission of giardiasis and the life cycle of many protists. Encystation for Giardia lamblia involves the assembly of a protective cyst wall via sequential production, trafficking, and secretion of cyst wall material. However, the regulatory pathways that coordinate cargo maturation and secretion remain unknown. Here, we asked whether the signaling activities of G. lamblia’s single Rho family GTPase, GlRac, might have a regulatory role in the encystation process. We show that GlRac localizes to endomembranes and its signaling activities regulate the production of cyst wall protein 1 (CWP1), the maturation of encystation-specific vesicles (ESVs), and secretion of CWP1. We also show that secreted CWP1 is available for the development of cysts at the population level, a finding that in part could explain why Giardia encystation proceeds more efficiently at high cell densities.