Biomedical subjects

Alice Y Cheung

Publications and source records attributed to Alice Y Cheung.

12 recordsLinked to original sources

PubMed2006

Structural and functional compartmentalization in pollen tubes.

Eukaryotic cellular functions are achieved by concerted activities in the cytosol and functions compartmentalized in the nucleus and other membrane-bound organelles. Moreover, the cytosol and nucleoplasm are populated with mega molecular ensembles that are specialized for different metabolic and biochemical processes. Pollen tubes are unique plant cells with a dramatic growth polarity. Tube growth is restricted to the tip and is supported by a polarized cytoplasmic organization. The apical region of elongating pollen tubes is a domain occupied exclusively by transport vesicles to support the secretion and endocytic activity needed for the rapid cell expansion at the apex. Larger organelles are predominantly segregated to the cytoplasm distal to the subapical region. Underlying the organelle compartmentalization is an elaborate actin cytoskeleton with distinct structural and dynamics properties at the tip, in the subapical region, and in the cytoplasm subtending it. Cytoplasmic domains with differential ionic conditions and spatially restricted localization of molecules in pollen tubes may also be important for regulating the polar cell growth process. The polarized cellular organization in pollen tubes drives an extremely efficient cell growth process that is responsive to extracellular signals, including directional cues. It may be an amplified framework of the cytoplasmic architecture that supports growth in other plant cell types that involves considerably more subtle and transient differential cell expansion.

Cell Compartmentation↗

PubMed2006

RAC/ROP GTPases: 'hubs' for signal integration and diversification in plants.

RAC/ROP GTPases are a family of plant-specific signaling molecules solely representing the Ras and Rho family of Ras-related G proteins in plants. RAC/ROPs potentially interact with cell surface-associated signal perception apparatus for a broad range of extracellular stimuli, including hormones, pathogen elicitors and abiotic stress, and mediate diverse cellular pathways in response to these signals. They are also known to interact with multiple effectors, affecting cellular and biochemical systems that regulate actin dynamics, reactive oxygen species production, proteolysis, and gene expression. RAC/ROPs are, thus, ideally suited as integrators for multiple signals and as coordinators of diverse cellular pathways to control growth, differentiation, development and defense responses. Recent findings that suggest how RAC/ROP signaling activity is regulated and how functional specificity can be achieved are discussed here.

Arabidopsis Proteins↗

PubMed2005

Rab11 GTPase-regulated membrane trafficking is crucial for tip-focused pollen tube growth in tobacco.

Pollen tube growth is a polarized growth process whereby the tip-growing tubes elongate within the female reproductive tissues to deliver sperm cells to the ovules for fertilization. Efficient and regulated membrane trafficking activity incorporates membrane and deposits cell wall molecules at the tube apex and is believed to underlie rapid and focused growth at the pollen tube tip. Rab GTPases, key regulators of membrane trafficking, are candidates for important roles in regulating pollen tube growth. We show that a green fluorescent protein-tagged Nicotiana tabacum pollen-expressed Rab11b is localized predominantly to an inverted cone-shaped region in the pollen tube tip that is almost exclusively occupied by transport vesicles. Altering Rab11 activity by expressing either a constitutive active or a dominant negative variant of Rab11b in pollen resulted in reduced tube growth rate, meandering pollen tubes, and reduced male fertility. These mutant GTPases also inhibited targeting of exocytic and recycled vesicles to the pollen tube inverted cone region and compromised the delivery of secretory and cell wall proteins to the extracellular matrix. Properly regulated Rab11 GTPase activity is therefore essential for tip-focused membrane trafficking and growth at the pollen tube apex and is pivotal to reproductive success.

Actins↗

PubMed2005

RAC GTPases in tobacco and Arabidopsis mediate auxin-induced formation of proteolytically active nuclear protein bodies that contain AUX/IAA proteins.

Auxin signaling relies on ubiquitin ligase SCF(TIR1)-mediated 26S proteasome-dependent proteolysis of a large family of short-lived transcription regulators, auxin/indole acetic acid (Aux/IAA), resulting in the derepression of auxin-responsive genes. We have shown previously that a subset of Rac GTPases is activated by auxin, and they in turn stimulate auxin-responsive gene expression. We show here that increasing Rac signaling activity promotes Aux/IAA degradation, whereas downregulating that activity results in the reduction of auxin-accelerated Aux/IAA proteolysis. Observations reported here reveal a novel function for these Rac GTPases as regulators for ubiquitin/26S proteasome-mediated proteolysis and further consolidate their role in auxin signaling. Moreover, our study reveals a cellular process whereby auxin induces and Rac GTPases mediate the recruitment of nucleoplasmic Aux/IAAs into proteolytically active nuclear protein bodies, into which components of the SCF(TIR1), COP9 signalosome, and 26S proteasome are also recruited.

Arabidopsis Proteins↗

PubMed2005

Pectin methylesterase, a regulator of pollen tube growth.

The apical wall of growing pollen tubes must be strong enough to withstand the internal turgor pressure, but plastic enough to allow the incorporation of new membrane and cell wall material to support polarized tip growth. These essential rheological properties appear to be controlled by pectins, which constitute the principal component of the apical cell wall. Pectins are secreted as methylesters and subsequently deesterified by the enzyme pectin methylesterase (PME) in a process that exposes acidic residues. These carboxyls can be cross-linked by calcium, which structurally rigidifies the cell wall. Here, we examine the role of PME in cell elongation and the regulation of its secretion and enzymatic activity. Application of an exogenous PME induces thickening of the apical cell wall and inhibits pollen tube growth. Screening a Nicotiana tabacum pollen cDNA library yielded a pollen-specific PME, NtPPME1, containing a pre-region and a pro-region. Expression studies with green fluorescent protein fusion proteins show that the pro-region participates in the correct targeting of the mature PME. Results from in vitro growth analysis and immunolocalization studies using antipectin antibodies (JIM5 and JIM7) provide support for the idea that the pro-region acts as an intracellular inhibitor of PME activity, thereby preventing premature deesterification of pectins. In addition to providing experimental data that help resolve the significance and function of the pro-region, our results give insight into the mechanism by which PME and its pro-region regulate the cell wall dynamics of growing pollen tubes.

Arabidopsis↗

PubMed2005

Molecular analysis of the interaction between cardosin A and phospholipase D(alpha). Identification of RGD/KGE sequences as binding motifs for C2 domains.

Here we report the identification of phospholipase Dalpha as a cardosin A-binding protein. The interaction was confirmed by coimmunoprecipitation studies and pull-down assays. To investigate the structural and molecular determinants involved in the interaction, pull-down assays with cardosin A and various glutathione S-transferase-fused phospholipase Dalpha constructs were performed. Results revealed that the C2 domain of phospholipase Dalpha contains the cardosin A-binding activity. Further assays with mutated recombinant forms of cardosin A showed that the RGD motif as well as the unprecedented KGE motif, which is structurally and charge-wise very similar to RGD, are indispensable for the interaction. Taken together our results indicate that the C2 domain of plant phospholipase Dalpha can act as a cardosin A-binding domain and suggest that plant C2 domains may have an additional role as RGD/KGE-recognition domains.

Amino Acid Sequence↗

PubMed2003

Overexpression of an Arabidopsis formin stimulates supernumerary actin cable formation from pollen tube cell membrane.

Formins, actin-nucleating proteins that stimulate the de novo polymerization of actin filaments, are important for diverse cellular and developmental processes, especially those dependent on polarity establishment. A subset of plant formins, referred to as group I, is distinct from formins from other species in having evolved a unique N-terminal structure with a signal peptide, a Pro-rich, potentially glycosylated extracellular domain, and a transmembrane domain. We show here that overexpression of the Arabidopsis formin AFH1 in pollen tubes induces the formation of arrays of actin cables that project into the cytoplasm from the cell membrane and that its N-terminal structure targets AFH1 to the cell membrane. Pollen tube elongation is a polar cell growth process dependent on an active and tightly regulated actin cytoskeleton. Slight increases in AFH1 stimulate growth, but its overexpression induces tube broadening, growth depolarization, and growth arrest in transformed pollen tubes. These results suggest that AFH1-regulated actin polymerization is important for the polar pollen cell growth process. Moreover, severe membrane deformation was observed in the apical region of tip-expanded, AFH1-overexpressing pollen tubes in which an abundance of AFH1-induced membrane-associated actin cables was evident. These observations suggest that regulated AFH1 activity at the cell surface is important for maintaining tip-focused cell membrane expansion for the polar extension of pollen tubes. The cell surface-located group-I formins may play the integrin-analogous role as mediators of external stimuli to the actin cytoskeleton, and AFH1 could be important for mediating extracellular signals from female tissues to elicit the proper pollen tube growth response during pollination.

Actins↗

PubMed2003

Regulation of pollen tube growth by Rac-like GTPases.

Plant Rac-like GTPases have been classified phylogenetically into two major groups-class I and class II. Several pollen-expressed class I Rac-like GTPases have been shown to be important regulators of polar pollen tube growth. The functional participation by some of the class I and all of the class II Arabidopsis Rac-like GTPases in pollen tube growth remains to be explored. It is shown that at least four members of the Arabidopsis Rac GTPase family are expressed in pollen, including a class II Rac, AtRac7. However, when over-expressed as fusion proteins with GFP, both pollen- and non-pollen-expressed AtRacs interfered with the normal pollen tube tip growth process. These observations suggest that these AtRacs share similar biochemical activities and may integrate into the pollen cellular machinery that regulates the polar tube growth process. Therefore, the functional contribution by individual Rac GTPase to the pollen tube growth process probably depends to a considerable extent on their expression characteristics in pollen. Among the Arabidopsis Racs, GFP-AtRac7 showed association with the cell membrane and Golgi bodies, a pattern distinct from all previously reported localization for other plant Racs. Over-expressing GFP-AtRac7 also induced the broadest spectrum of pollen tube growth defects, including pollen tubes that are bifurcated, with diverted growth trajectory or a ballooned tip. Transgenic plants with multiple copies of the chimeric Lat52-GFP-AtRac7 showed severely reduced seed set, probably many of these defective pollen tubes were arrested, or reduced in their growth rates that they did not arrive at the ovules while they were still receptive for fertilization. These observations substantiate the importance of Rac-like GTPases to sexual reproduction.

Arabidopsis↗

PubMed2003

Actin-depolymerizing factor mediates Rac/Rop GTPase-regulated pollen tube growth.

Pollen tube elongation is a rapid tip growth process that is driven by a dynamic actin cytoskeleton. A ubiquitous family of actin binding proteins, actin-depolymerizing factors (ADFs)/cofilins, bind to actin filaments, induce severing, enhance depolymerization from their slow-growing end, and are important for maintaining actin dynamics in vivo. ADFs/cofilins are regulated by multiple mechanisms, among which Rho small GTPase-activated phosphorylation at a terminal region Ser residue plays an important role in regulating their actin binding and depolymerizing activity, affecting actin reorganization. We have shown previously that a tobacco pollen-specific ADF, NtADF1, is important for maintaining normal pollen tube actin cytoskeleton organization and growth. Here, we show that tobacco pollen grains accumulate phosphorylated and nonphosphorylated forms of ADFs, suggesting that phosphorylation could be a regulatory mechanism for their activity. In plants, Rho-related Rac/Rop GTPases have been shown to be important regulators for pollen tube growth. Overexpression of Rac/Rop GTPases converts polar growth into isotropic growth, resulting in pollen tubes with ballooned tips and a disrupted actin cytoskeleton. Using the Rac/Rop GTPase-induced defective pollen tube phenotype as a functional assay, we show that overexpression of NtADF1 suppresses the ability of NtRac1, a tobacco Rac/Rop GTPase, to convert pollen tube tip growth to isotropic growth. This finding suggests that NtADF1 acts in a common pathway with NtRac1 to regulate pollen tube growth. A mutant form of NtADF1 with a nonphosphorylatable Ala substitution at its Ser-6 position [NtADF1(S6A)] shows increased activity, whereas the mutant NtADF1(S6D), which has a phospho-mimicking Asp substitution at the same position, shows reduced ability to counteract the effect of NtRac1. These observations suggest that phosphorylation at Ser-6 of NtADF1 could be important for its integration into the NtRac1 signaling pathway. Moreover, overexpression of NtRac1 diminishes the actin binding activity of green fluorescent protein (GFP)-NtADF1 but has little effect on the association of GFP-NtADF1(S6A) with actin cables in pollen tubes. Together, these observations suggest that NtRac1-activated activity regulates the actin binding and depolymerizing activity of NtADF1, probably via phosphorylation at Ser-6. This notion is further supported by the observation that overexpressing a constitutively active NtRac1 in transformed pollen grains significantly increases the ratio of phosphorylated to nonphosphorylated ADFs. Together, the observations reported here strongly support the idea that NtRac1 modulates NtADF1 activity through phosphorylation at Ser-6 to regulate actin dynamics.

Actin Depolymerizing Factors↗

PubMed2002

Rab2 GTPase regulates vesicle trafficking between the endoplasmic reticulum and the Golgi bodies and is important to pollen tube growth.

Pollen tube elongation depends on the secretion of large amounts of membrane and cell wall materials at the pollen tube tip to sustain rapid growth. A large family of RAS-related small GTPases, Rabs or Ypts, is known to regulate both anterograde and retrograde trafficking of transport vesicles between different endomembrane compartments and the plasma membrane in mammalian and yeast cells. Studies on the functional roles of analogous plant proteins are emerging. We report here that a tobacco pollen-predominant Rab2, NtRab2, functions in the secretory pathway between the endoplasmic reticulum and the Golgi in elongating pollen tubes. Green fluorescent protein-NtRab2 fusion protein localized to the Golgi bodies in elongating pollen tubes. Dominant-negative mutations in NtRab2 proteins inhibited their Golgi localization, blocked the delivery of Golgi-resident as well as plasmalemma and secreted proteins to their normal locations, and inhibited pollen tube growth. On the other hand, when green fluorescent protein-NtRab2 was over-expressed in transiently transformed leaf protoplasts and epidermal cells, in which NtRab2 mRNA have not been observed to accumulate to detectable levels, these proteins did not target efficiently to Golgi bodies. Together, these observations indicate that NtRab2 is important for trafficking between the endoplasmic reticulum and the Golgi bodies in pollen tubes and may be specialized to optimally support the high secretory demands in these tip growth cells.

Biological Transport↗

PubMed2002

The regulation of actin organization by actin-depolymerizing factor in elongating pollen tubes.

Pollen tube elongation is a polarized cell growth process that transports the male gametes from the stigma to the ovary for fertilization inside the ovules. Actomyosin-driven intracellular trafficking and active actin remodeling in the apical and subapical regions of pollen tubes are both important aspects of this rapid tip growth process. Actin-depolymerizing factor (ADF) and cofilin are actin binding proteins that enhance the depolymerization of microfilaments at their minus, or slow-growing, ends. A pollen-specific ADF from tobacco, NtADF1, was used to dissect the role of ADF in pollen tube growth. Overexpression of NtADF1 resulted in the reduction of fine, axially oriented actin cables in transformed pollen tubes and in the inhibition of pollen tube growth in a dose-dependent manner. Thus, the proper regulation of actin turnover by NtADF1 is critical for pollen tube growth. When expressed at a moderate level in pollen tubes elongating in in vitro cultures, green fluorescent protein (GFP)-tagged NtADF1 (GFP-NtADF1) associated predominantly with a subapical actin mesh composed of short actin filaments and with long actin cables in the shank. Similar labeling patterns were observed for GFP-NtADF1-expressing pollen tubes elongating within the pistil. A Ser-6-to-Asp conversion abolished the interaction between NtADF1 and F-actin in elongating pollen tubes and reduced its inhibitory effect on pollen tube growth significantly, suggesting that phosphorylation at Ser-6 may be a prominent regulatory mechanism for this pollen ADF. As with some ADF/cofilin, the in vitro actin-depolymerizing activity of recombinant NtADF1 was enhanced by slightly alkaline conditions. Because a pH gradient is known to exist in the apical region of elongating pollen tubes, it seems plausible that the in vivo actin-depolymerizing activity of NtADF1, and thus its contribution to actin dynamics, may be regulated spatially by differential H(+) concentrations in the apical region of elongating pollen tubes.

Actins↗

PubMed2002

Plant Rac-like GTPases are activated by auxin and mediate auxin-responsive gene expression.

The auxin indole-3-acetic acid is a key plant hormone essential for a broad range of growth and developmental processes. Here, we show that auxin activates Rac-like GTPases (referred to as Rac/Rop GTPases), and they in turn stimulate auxin-responsive gene expression. In particular, we show that overexpressing a wild-type tobacco Rac/Rop GTPase, NtRac1, and its constitutively active mutant form activates auxin-responsive gene expression. On the other hand, overexpressing dominant-negative NtRac1 and Rac-negative regulators, or reducing the endogenous NtRac1 level, suppresses auxin-induced gene expression. Furthermore, overexpression of NtRac1 activity or suppression of its expression in transgenic seedlings induces phenotypes that are similar to auxin-related defects. Together, our results show that a subset of plant Rac/Rop GTPases functions in mediating the auxin signal to downstream responsive genes.

Arabidopsis Proteins↗