Biomedical subjects

Patrick J Hussey

Publications and source records attributed to Patrick J Hussey.

At least 19 recordsLinked to original sources

PubMed2006

The POLARIS peptide of Arabidopsis regulates auxin transport and root growth via effects on ethylene signaling.

The rate and plane of cell division and anisotropic cell growth are critical for plant development and are regulated by diverse mechanisms involving several hormone signaling pathways. Little is known about peptide signaling in plant growth; however, Arabidopsis thaliana POLARIS (PLS), encoding a 36-amino acid peptide, is required for correct root growth and vascular development. Mutational analysis implicates a role for the peptide in hormone responses, but the basis of PLS action is obscure. Using the Arabidopsis root as a model to study PLS action in plant development, we discovered a link between PLS, ethylene signaling, auxin homeostasis, and microtubule cytoskeleton dynamics. Mutation of PLS results in an enhanced ethylene-response phenotype, defective auxin transport and homeostasis, and altered microtubule sensitivity to inhibitors. These defects, along with the short-root phenotype, are suppressed by genetic and pharmacological inhibition of ethylene action. PLS expression is repressed by ethylene and induced by auxin. Our results suggest a mechanism whereby PLS negatively regulates ethylene responses to modulate cell division and expansion via downstream effects on microtubule cytoskeleton dynamics and auxin signaling, thereby influencing root growth and lateral root development. This mechanism involves a regulatory loop of auxin-ethylene interactions.

Amino Acids, Cyclic↗

PubMed2006

Oscillatory increases in alkalinity anticipate growth and may regulate actin dynamics in pollen tubes of lily.

Lily (Lilium formosanum or Lilium longiflorum) pollen tubes, microinjected with a low concentration of the pH-sensitive dye bis-carboxyethyl carboxyfluorescein dextran, show oscillating pH changes in their apical domain relative to growth. An increase in pH in the apex precedes the fastest growth velocities, whereas a decline follows growth, suggesting a possible relationship between alkalinity and cell extension. A target for pH may be the actin cytoskeleton, because the apical cortical actin fringe resides in the same region as the alkaline band in lily pollen tubes and elongation requires actin polymerization. A pH-sensitive actin binding protein, actin-depolymerizing factor (ADF), together with actin-interacting protein (AIP) localize to the cortical actin fringe region. Modifying intracellular pH leads to reorganization of the actin cytoskeleton, especially in the apical domain. Acidification causes actin filament destabilization and inhibits growth by 80%. Upon complete growth inhibition, the actin fringe is the first actin cytoskeleton component to disappear. We propose that during normal growth, the pH increase in the alkaline band stimulates the fragmenting activity of ADF/AIP, which in turn generates more sites for actin polymerization. Increased actin polymerization supports faster growth rates and a proton influx, which inactivates ADF/AIP, decreases actin polymerization, and retards growth. As pH stabilizes and increases, the activity of ADF/AIP again increases, repeating the cycle of events.

Actin Cytoskeleton↗

PubMed2006

Control of the AtMAP65-1 interaction with microtubules through the cell cycle.

Cell division depends on the fine control of both microtubule dynamics and microtubule organisation. The microtubule bundling protein MAP65 is a ;midzone MAP' essential for the integrity of the anaphase spindle and cell division. Arabidopsis thaliana MAP65-1 (AtMAP65-1) binds and bundles microtubules by forming 25 nm cross-bridges. Moreover, as AtMAP65-1 bundles microtubules in interphase, anaphase and telophase but does not bind microtubules in prophase or metaphase, its activity through the cell cycle must be under tight control. Here we show that AtMAP65-1 is hyperphosphorylated during prometaphase and metaphase and that CDK and MAPK are involved in this phosphorylation. This phosphorylation inhibits AtMAP65-1 activity. Expression of non-phosphorylatable AtMAP65-1 has a negative effect on mitotic progression resulting in excessive accumulation of microtubules in the metaphase spindle midzone causing a delay in mitosis. We conclude that normal metaphase spindle organisation and the transition to anaphase is dependent on inactivation of AtMAP65-1.

Arabidopsis↗

PubMed2006

Control of the actin cytoskeleton in plant cell growth.

Plant cells grow through increases in volume and cell wall surface area. The mature morphology of a plant cell is a product of the differential rates of expansion between neighboring zones of the cell wall during this process. Filamentous actin arrays are associated with plant cell growth, and the activity of actin-binding proteins is proving to be essential for proper cell morphogenesis. Actin-nucleating proteins participate in cell expansion and cell plate formation whereas the recycling of actin monomers is required to maintain actin dynamics and controlled growth. Coordination of actin-binding protein activity and other aspects of cytoskeletal behavior during cell development maintains cohesive cell expansion. Emerging plant signaling networks are proving to be powerful regulators of morphology-shaping cytoskeletal activity, and in this review we highlight current research in actin network regulation.

Actins↗

PubMed2005

A divergent cellular role for the FUSED kinase family in the plant-specific cytokinetic phragmoplast.

The FUSED (FU) Ser/Thr protein kinase family has a key role in the hedgehog signaling pathway known to control cell proliferation and patterning in fruit flies and humans . The genomes of Arabidopsis thaliana and rice each encode a single Fu ortholog, but their role is unknown. Here, we show that cytokinesis-defective mutants, which we named two-in-one (tio), result from mutations in Arabidopsis Fu. Phenotypic analysis of tio mutants reveals an essential role for TIO in conventional modes of cytokinesis in plant meristems and during male gametogenesis. TIO also has a key role in nonconventional modes of cytokinesis (cellularization) during female gametogenesis. We demonstrate that TIO is tightly localized to the midline of the nascent phragmoplast and remains associated with the expanding phragmoplast ring. These data reveal the evolution of a divergent role for the Fu kinase family as an essential phragmoplast-associated protein that functions in different cell type-specific modes of cytokinesis in plants.

Arabidopsis↗

PubMed2005

Dynamic interaction of NtMAP65-1a with microtubules in vivo.

Plant microtubules are intrinsically more dynamic than those from animals. We know little about the dynamics of the interaction of plant microtubule-associated proteins (MAPs) with microtubules. Here, we have used tobacco and Arabidopsis MAPs with relative molecular mass 65 kDa (NtMAP65-1a and AtMAP65-1), to study their interaction with microtubules in vivo. Using fluorescence recovery after photobleaching we report that the turnover of both NtMAP65-1a and AtMAP65-1 bound to microtubules is four- to fivefold faster than microtubule treadmilling (13 seconds compared with 56 seconds, respectively) and that the replacement of NtMAP65-1a on microtubules is by random association rather than by translocation along microtubules. MAP65 will only bind polymerised microtubules and not its component tubulin dimers. The turnover of NtMAP65-1a and AtMAP65-1 on microtubules is similar in the interphase cortical array, the preprophase band and the phragmoplast, strongly suggesting that their role in these arrays is the same. NtMAP65-1a and AtMAP65-1 are not observed to bind microtubules in the metaphase spindle and their rate of recovery is consistent with their cytoplasmic localisation. In addition, the dramatic reappearance of NtMAP65-1a on microtubules at the spindle midzone in anaphase B suggests that NtMAP65-1a is controlled post-translationally. We conclude that the dynamic properties of these MAPs in vivo taken together with the fact that they have been shown not to effect microtubule polymerisation in vitro, makes them ideally suited to a role in crossbridging microtubules that need to retain spatial organisation in rapidly reorganising microtubule arrays.

Arabidopsis↗

PubMed2005

A Rab-E GTPase mutant acts downstream of the Rab-D subclass in biosynthetic membrane traffic to the plasma membrane in tobacco leaf epidermis.

The function of the Rab-E subclass of plant Rab GTPases in membrane traffic was investigated using a dominant-inhibitory mutant (RAB-E1(d)[NI]) of Arabidopsis thaliana RAB-E1(d) and in vivo imaging approaches that have been used to characterize similar mutants in the plant Rab-D2 and Rab-F2 subclasses. RAB-E1(d)[NI] inhibited the transport of a secreted green fluorescent protein marker, secGFP, but in contrast with dominant-inhibitory RAB-D2 or RAB-F2 mutants, it did not affect the transport of Golgi or vacuolar markers. Quantitative imaging revealed that RAB-E1(d)[NI] caused less intracellular secGFP accumulation than RAB-D2(a)[NI], a dominant-inhibitory mutant of a member of the Arabidopsis Rab-D2 subclass. Furthermore, whereas RAB-D2(a)[NI] caused secGFP to accumulate exclusively in the endoplasmic reticulum, RAB-E1(d)[NI] caused secGFP to accumulate additionally in the Golgi apparatus and a prevacuolar compartment that could be labeled by FM4-64 and yellow fluorescent protein (YFP)-tagged Arabidopsis RAB-F2(b). Using the vacuolar protease inhibitor E64-d, it was shown that some secGFP was transported to the vacuole in control cells and in the presence of RAB-E1(d)[NI]. Consistent with the hypothesis that secGFP carries a weak vacuolar-sorting determinant, it was shown that a secreted form of DsRed reaches the apoplast without appearing in the prevacuolar compartment. When fused to RAB-E1(d), YFP was targeted specifically to the Golgi via a saturable nucleotide- and prenylation-dependent mechanism but was never observed on the prevacuolar compartment. We propose that RAB-E1(d)[NI] inhibits the secretory pathway at or after the Golgi, causing an accumulation of secGFP in the upstream compartments and an increase in the quantity of secGFP that enters the vacuolar pathway.

Bacterial Proteins↗

PubMed2005

Arp2/3 and SCAR: plants move to the fore.

The actin-nucleating Arp2/3 complex is essential for life in yeast and animals, but not in plants, in which mutants of Arp2/3 complex components show relatively minor developmental abnormalities. Animal cells control the activity of the Arp2/3 complex through the suppressor of cyclic AMP receptor (SCAR) complex to achieve cell motility. Amazingly, plants have also retained the SCAR cell-motility pathway, and now provide a unique model for the study of new aspects of SCAR function in the absence of cell motility.

Actin-Related Protein 2-3 Complex↗

PubMed2005

Arabidopsis group Ie formins localize to specific cell membrane domains, interact with actin-binding proteins and cause defects in cell expansion upon aberrant expression.

The closely related proteins AtFH4 and AtFH8 represent the group Ie clade of Arabidopsis formin homologues. The subcellular localization of these proteins and their ability to affect the actin cytoskeleton were examined. AtFH4 protein activity was identified using fluorimetric techniques. Interactions between Arabidopsis profilin isoforms and AtFH4 were assayed in vitro and in vivo using pull-down assays and yeast-2-hybrid. The subcellular localization of group Ie formins was observed with indirect immunofluorescence (AtFH4) and an ethanol-inducible green fluorescent protein (GFP) fusion construct (AtFH8). AtFH4 protein affected actin dynamics in vitro, and yeast-2-hybrid assays suggested isoform-specific interactions with the actin-binding protein profilin in vivo. Indirect immunofluorescence showed that AtFH4 localized specifically to the cell membrane at borders between adjoining cells. Expression of an AtFH8 fusion protein resulted in GFP localization to cell membrane zones, similar to AtFH4. Furthermore, aberrant expression of AtFH8 resulted in the inhibition of root hair elongation. Taken together, these data suggest that the group Ie formins act with profilin to regulate actin polymerization at specific sites associated with the cell membrane.

Actins↗

PubMed2004

Green fluorescent protein-mTalin causes defects in actin organization and cell expansion in Arabidopsis and inhibits actin depolymerizing factor's actin depolymerizing activity in vitro.

Expression of green fluorescent protein (GFP) linked to an actin binding domain is a commonly used method for live cell imaging of the actin cytoskeleton. One of these chimeric proteins is GFP-mTalin (GFP fused to the actin binding domain of mouse talin). Although it has been demonstrated that GFP-mTalin colocalizes with the actin cytoskeleton, its effect on actin dynamics and cell expansion has not been studied in detail. We created Arabidopsis (Arabidopsis thaliana) plants harboring alcohol inducible GFP-mTalin constructs to assess the effect of GFP-mTalin expression in vivo. We focused on the growing root hair as this is a model cell for studying cell expansion and root hair tip growth that requires a highly dynamic and polar actin cytoskeleton. We show that alcohol inducible expression of GFP-mTalin in root hairs causes severe defects in actin organization, resulting in either the termination of growth, cell death, and/or changes in cell shape. Fluorescence recovery after photobleaching experiments demonstrate that the interaction of GFP-mTalin and actin filaments is highly dynamic. To assess how GFP-mTalin affects actin dynamics we performed cosedimentation assays of GFP-mTalin with actin on its own or in the presence of the actin modulating protein, actin depolymerizing factor. We show that that GFP-mTalin does not affect actin polymerization but that it does inhibit the actin depolymerizing activity of actin depolymerizing factor. These observations demonstrate that GFP-mTalin can affect cell expansion, actin organization, and the interaction of actin binding proteins with actin.

Actin Depolymerizing Factors↗

PubMed2004

Arabidopsis NAP1 is essential for Arp2/3-dependent trichome morphogenesis.

The dynamic nature of the eukaryotic actin cytoskeleton is essential for the locomotion of animal cells and the morphogenesis of plant and fungal cells. The F-actin nucleating/branching activity of the Arp2/3 complex is a key function for all of these processes. The SCAR/WAVE family represents a group of Arp2/3 activators that are associated with lamellipodia formation. A protein complex of PIR121, NAP1, ABI, and HSPC300 is required for SCAR regulation by cell signaling pathways, but the exact nature of this interaction is controversial and represents a continually evolving model. The mechanism originally proposed was of a SCAR trans repressing complex supported by evidence from in vitro experiments. This model was reinforced by genetic studies in the Drosophila central nervous system and Dictyostelium, where the knockout of certain SCAR-complex components leads to excessive SCAR-mediated actin polymerization. Conflicting data have steadily accumulated from animal tissue culture experiments suggesting that the complex activates rather than represses in vivo SCAR activity. Recent biochemical evidence supports the SCAR-complex activator model. Here, we show that genetic observations in Arabidopsis are compatible with an activation model and provide one potential mechanism for the regulation of the newly identified Arabidopsis Arp2/3 complex.

Actin-Related Protein 2↗

PubMed2004

The Arabidopsis microtubule-associated protein AtMAP65-1: molecular analysis of its microtubule bundling activity.

The 65-kD microtubule-associated protein (MAP65) family is a family of plant microtubule-bundling proteins. Functional analysis is complicated by the heterogeneity within this family: there are nine MAP65 genes in Arabidopsis thaliana, AtMAP65-1 to AtMAP65-9. To begin the functional dissection of the Arabidopsis MAP65 proteins, we have concentrated on a single isoform, AtMAP65-1, and examined its effect on the dynamics of mammalian microtubules. We show that recombinant AtMAP65-1 does not promote polymerization and does not stabilize microtubules against cold-induced microtubule depolymerization. However, we show that it does induce microtubule bundling in vitro and that this protein forms 25-nm cross-bridges between microtubules. We further demonstrate that the microtubule binding region resides in the C-terminal half of the protein and that Ala409 and Ala420 are essential for the interaction with microtubules. Ala420 is a conserved amino acid in the AtMAP65 family and is mutated to Val in the cytokinesis-defective mutant pleiade-4 of the AtMAP65-3/PLEIADE gene. We show that AtMAP65-1 can form dimers and that a region in the N terminus is responsible for this activity. Neither the microtubule binding region nor the dimerization region alone could induce microtubule bundling, strongly suggesting that dimerization is necessary to produce the microtubule cross-bridges. In vivo, AtMAP65-1 is ubiquitously expressed both during the cell cycle and in all plant organs and tissues with the exception of anthers and petals. Moreover, using an antiserum raised to AtMAP65-1, we show that AtMAP65-1 binds microtubules at specific stages of the cell cycle.

Amino Acid Sequence↗

PubMed2004

Arabidopsis homologues of the autophagy protein Atg8 are a novel family of microtubule binding proteins.

Autophagy is the non-selective transport of proteins and superfluous organelles destined for degradation to the vacuole in fungae, or the lysosome in animal cells. Some of the genes encoding components of the autophagy pathway are conserved in plants, and here we show that Arabidopsis homologues of yeast Atg8 (Apg8/Aut7) and Atg4 (Apg4/Aut2) partially complement the yeast deletion strains. The yeast double mutant, a deletion strain with respect to both Atg8 and Atg4, could not be complemented by Arabidopsis Atg8, indicating that Arabidopsis Atg8 requires Atg4 for its function. Moreover, Arabidopsis Atg8 and Arabidopsis Atg4 interact directly in a two-hybrid assay. Interestingly, Atg8 shows significant homology with the microtubule binding light chain 3 of MAP1A and B, and here we show that Arabidopsis Atg8 binds microtubules. Our results demonstrate that a principle component of the autophagic pathway in plants is similar to that in yeast and we suggest that microtubule binding plays a role in this process.

Arabidopsis↗

PubMed2004

The plant microtubule-associated protein AtMAP65-3/PLE is essential for cytokinetic phragmoplast function.

Directional cell expansion in interphase and nuclear and cell division in M-phase are mediated by four microtubule arrays, three of which are unique to plants: the interphase array, the preprophase band, and the phragmoplast. The plant microtubule-associated protein MAP65 has been identified as a key structural component in these arrays. The Arabidopsis genome has nine MAP65 genes, and here we show that one, AtMAP65-3/PLE, locates only to the mitotic arrays and is essential for cytokinesis. The Arabidopsis pleiade (ple) alleles are single recessive mutations, and we show that these mutations are in the AtMAP65-3 gene. Moreover, these mutations cause C-terminal truncations that abolish microtubule binding. In the ple mutants the anaphase spindle is normal, and the cytokinetic phragmoplast can form but is distorted; not only is it wider, but the midline, the region where oppositely oriented microtubules overlap, is unusually expanded. Here we present data that demonstrate an essential role for AtMAP65-3/PLE in cytokinesis in plant cells.

Alleles↗

PubMed2004

The actin-interacting protein AIP1 is essential for actin organization and plant development.

Cell division, growth, and cytoplasmic organization require a dynamic actin cytoskeleton. The filamentous actin (F-actin) network is regulated by actin binding proteins that modulate actin dynamics. These actin binding proteins often have cooperative interactions. In particular, actin interacting protein 1 (AIP1) is capable of capping F-actin and enhancing the activity of the small actin modulating protein, actin depolymerising factor (ADF) in vitro. Here, we analyze the effect of the inducible expression of AIP1 RNAi in Arabidopsis plants to assess AIP1s role in vivo. In intercalary growing cells, the normal actin organization is disrupted, and thick bundles of actin appear in the cytoplasm. Moreover, in root hairs, there is the unusual appearance of actin cables ramifying the root hair tip. We suggest that the reduction in AIP1 results in a decrease in F-actin turnover and the promotion of actin bundling. This distortion of the actin cytoskeleton causes severe plant developmental abnormalities. After induction of the Arabidopis RNAi lines, the cells in the leaves, roots, and shoots fail to expand normally, and in the severest phenotypes, the plants die. Our data suggest that AIP1 is essential for the normal functioning of the actin cytoskeleton in plant development.

Actins↗

PubMed2003

Identification of a MAP65 isoform involved in directional expansion of plant cells.

MAP65 comprises a multigene family specific to plants. To see which isoform is utilised for the unique mechanism of cell expansion, uncomplicated by division structures, carrot cells were deprived of auxin whereupon they stopped dividing and elongated instead. During elongation, a MAP65 protein triplet reduced to a single band. Mass spectrometric analysis demonstrated that this corresponded to a single carrot cDNA; it also corresponded to the major protein previously shown to form filamentous cross-bridges between microtubules in vitro. This MAP65 isoform is concluded to have a major role in establishing the parallel microtubule arrays characteristic of cells undergoing directional expansion.

Amino Acid Sequence↗

PubMed2003

Arp2/3 and 'the shape of things to come'.

The shape of plant cells is reliant upon an organised actin network. A series of recent publications have emphasised the central role played by the actin-nucleating Arp2/3 complex in plant growth. In animals and fungi, the Arp2/3 complex influences cell motility and morphogenesis through its ability to nucleate actin filaments locally in response to cell signalling pathways. There are potential parallels in the action and control of the Arp2/3 complex in plants and animals.

Actin-Related Protein 2↗

PubMed2003

Re-organisation of the cytoskeleton during developmental programmed cell death in Picea abies embryos.

Cell and tissue patterning in plant embryo development is well documented. Moreover, it has recently been shown that successful embryogenesis is reliant on programmed cell death (PCD). The cytoskeleton governs cell morphogenesis. However, surprisingly little is known about the role of the cytoskeleton in plant embryogenesis and associated PCD. We have used the gymnosperm, Picea abies, somatic embryogenesis model system to address this question. Formation of the apical-basal embryonic pattern in P. abies proceeds through the establishment of three major cell types: the meristematic cells of the embryonal mass on one pole and the terminally differentiated suspensor cells on the other, separated by the embryonal tube cells. The organisation of microtubules and F-actin changes successively from the embryonal mass towards the distal end of the embryo suspensor. The microtubule arrays appear normal in the embryonal mass cells, but the microtubule network is partially disorganised in the embryonal tube cells and the microtubules disrupted in the suspensor cells. In the same embryos, the microtubule-associated protein, MAP-65, is bound only to organised microtubules. In contrast, in a developmentally arrested cell line, which is incapable of normal embryonic pattern formation, MAP-65 does not bind the cortical microtubules and we suggest that this is a criterion for proembryogenic masses (PEMs) to passage into early embryogeny. In embryos, the organisation of F-actin gradually changes from a fine network in the embryonal mass cells to thick cables in the suspensor cells in which the microtubule network is completely degraded. F-actin de-polymerisation drugs abolish normal embryonic pattern formation and associated PCD in the suspensor, strongly suggesting that the actin network is vital in this PCD pathway.

Actins↗