efficient-polyethylene-glycol-peg-mediated-transformation-moss

Efficient Polyethylene Glycol (PEG) Mediated Transformation of the Moss <em>Physcomitrella patens</em>

A simple and efficient method to transform Physcomitrella pantens protoplasts is described. This method is adapted from protocols for Physocmitrella ...

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.

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

RAB2 GTPase reguliert Vesikeltransport zwischen dem endoplasmatischen Retikulum und Golgi-K&#xF6;rper und ist wichtig, Pollenschlauchwachstum.

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.

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

Die Regulation der Aktin-Organisation, die von Aktin-Depolymerisation Faktor verl&#xE4;ngert Pollenschl&#xE4;uche.

In many cases, actin filaments are arranged into bundles and serve as tracks for cytoplasmic streaming in plant cells. We have isolated an actin-filament bundling protein, which is composed of 115-kDa polypeptide (P-115-ABP), from the germinating pollen of lily, Lilium longiflorum [Nakayasu et al. (1998) BIOCHEM: Biophys. Res. Commun. 249: 61]. P-115-ABP shared similar antigenicity with a plant 135-kDa actin-filament bundling protein (P-135-ABP), a plant homologue of villin. A full-length cDNA clone (ABP115; accession no. AB097407) was isolated from an expression cDNA library of lily pollen by immuno-screening using antisera against P-115-ABP and P-135-ABP. The amino acid sequence of P-115-ABP deduced from this clone showed high homology with those of P-135-ABP and four villin isoforms of Arabidopsis thaliana (AtVLN1, AtVLN2, AtVLN3 and AtVLN4), especially AtVLN4, indicating that P-115-ABP can also be classified as a plant villin. The P-115-ABP isolated biochemically from the germinating lily pollen was able to arrange F-actin filaments with uniform polarity into bundles and this bundling activity was suppressed by Ca2+-calmodulin (CaM), similar to the actin-filament bundling properties of P-135-ABP. The P-115-ABP type of plant villin was widely distributed in plant cells, from algae to land plants. In root hair cells of Hydrocharis dubia, this type of plant villin was co-localized with actin-filament bundles in the transvacuolar strands and the sub-cortical regions. Microinjection of the antiserum against P-115-ABP into living root hair cells caused the disappearance of transvaculor strands and alteration of the route of cytoplasmic streaming. In internodal cells of Chara corallina in which the P-135-ABP type of plant villin is lacking, the P-115-ABP type showed co-localization with actin-filament cables anchored on the intracellular surface of chloroplasts. These results indicated that plant villins are widely distributed and involved in the organization of actin filaments into bundles throughout the plant kingdom.

Plant 115-kDa actin-filament bundling protein, P-115-ABP, is a homologue of plant villin and is widely distributed in cells.

Pflanze 115-kDa Aktin-Filament-B&#xFC;ndeln Protein, P-115-ABD, ist ein Homolog von Pflanzenschutzmitteln Villin und wird in Zellen verteilt ist.

Previously, we have shown that excess profilin inhibits pollen tube growth at significantly lower concentrations than it blocks cytoplasmic streaming. To elucidate the mechanism by which profilin achieves this function, we have employed mutant profilins from Schizosaccharomyces pombe [J. Lu and T.D. Pollard (2001) Mol Biol Cell 12:1161-1175], which have defects in actin-binding, ability to inhibit polymerization, and poly- l-proline (PLP)-binding. Using Lilium longiflorum L. pollen and S. pombe profilins as wild-type (wt) standards, mutant profilins have been injected into pollen tubes of Lilium, and examined for their effects on growth rate and cell morphology. Our results show that mutant Y5D (68% actin-binding; 1.1% PLP-binding) is indistinguishable from wt-standard profilins. However mutant K81F (2.7% actin-binding; 77% PLP-binding) and especially mutant K67E (<1% actin-binding; 100% PLP-binding) are significantly less effective than wt-standard profilins in their ability to inhibit pollen tube growth. PLP also inhibits pollen tube growth. However, PLP is not different from K67E/PLP combined, which has no actin-binding, suggesting that PLP does not function by binding to profilin. In addition, there are differences in the morphology and F-actin organization in cells injected with PLP versus wt-profilin. Whereas wt-profilin causes a fragmentation and marked reduction in the amount of F-actin [L. Vidali et al. (2001) Mol Biol Cell 12:2534-2545], PLP generates an extensive disorganization without any apparent reduction in the amount of F-actin. We conclude that along with actin-binding activity of profilin, PLP-containing proteins also participate in the growth control process, and can do so independently of binding to profilin.

Profilin inhibits pollen tube growth through actin-binding, but not poly-L-proline-binding.

Profilin hemmt Pollenschlauchwachstum durch Aktin-bindende, aber nicht Poly-L-Prolin-Bindung.

Previous studies of Rac1 in fibroblasts have used dominant negative constructs, which may have nonspecific effects. We used a conditional Rac1 allele to critically examine Rac1 function in mouse fibroblasts. Lack of Rac1 had dramatic effects on nonconfluent cells, which were elongated and had extensive blebbing, but no lamellipodia or ruffle formation. However, Rac1-null fibroblasts translocated using pseudopodia-like protrusions without lamellipodia, migrating toward a platelet-derived growth factor (PDGF) gradient as efficiently as their wild-type counterparts. Rac1-null fibroblasts closed wounds in vitro and spread on a fibronectin substrate, although at a slower rate than wild-type cells. However, Rac1-null cells were markedly impaired in proliferation, with a defect in G1 to S transition, although they were capable of surviving in culture for more than 2 wk. These results refine our understanding of the functions of Rac1, indicate that lamellipodia formation is not required for cell motility, and show that PDGF-induced chemotaxis can occur in the absence of both lamellipodia and Rac1.

Rac1-null mouse embryonic fibroblasts are motile and respond to platelet-derived growth factor.

Fr&uuml;here Studien der Rac1 in Fibroblasten haben dominante negative Konstrukte, benutzt, die unspezifische Wirkungen haben k&ouml;nnen. Wir verwendeten eine bedingte Rac1-Allel, um Rac1-Funktion in Maus-Fibroblasten kritisch zu pr&uuml;fen. Mangelnde Rac1 hatte dramatische Auswirkungen auf nonconfluent Zellen, die waren l&auml;nglich und hatte umfangreiche Blebbing, aber keine Lamellipodium oder R&uuml;schen-Formation. Rac1-Null Fibroblasten transloziert jedoch mit Pseudopodien-artigen Vorspr&uuml;ngen ohne Lamellipodium, so effizient wie ihre Gegenst&uuml;cke Wildtyp gegen&uuml;ber einem Platelet-derived Growth Factor (PDGF)-Verlauf migrieren. Rac1-Null Fibroblasten geschlossen Wunden in vitro und verteilt auf einem Substrat Fibronektin, obwohl in einem langsameren Tempo als Wild-Typ-Zellen. Jedoch wurden Rac1-Null-Zellen deutlich in Vermehrung, mit einem Defekt in G1 S-&Uuml;bergang, beeintr&auml;chtigt obwohl sie &uuml;berleben in der Kultur f&uuml;r mehr als 2 wk f&auml;hig waren. Diese Ergebnisse verfeinern unser Verst&auml;ndnis der Funktionen der Rac1, anzugeben, dass Lamellipodium Formation nicht erforderlich f&uuml;r Zelle Motilit&auml;t ist und zeigen, dass die PDGF-induzierter Chemotaxis in Abwesenheit von Lamellipodium und Rac1 auftreten kann.

Rac1-Null M&auml;useembryonalen sind beweglich und reagieren auf Platelet-derived Growth Factor.

Mutations in the human Filamin A (FLNA) gene disrupt neuronal migration to the cerebral cortex and cause cardiovascular defects. Complete loss of Flna in mice results in embryonic lethality with severe cardiac structural defects involving ventricles, atria, and outflow tracts, as well as widespread aberrant vascular patterning. Despite these widespread developmental defects, migration and motility of many cell types does not appear to be affected. Instead, Flna-null embryos display abnormal epithelial and endothelial organization and aberrant adherens junctions in developing blood vessels, heart, brain, and other tissues. Essential roles for FLNA in intercellular junctions provide a mechanism for the diverse developmental defects seen in patients with FLNA mutations.

Filamin A (FLNA) is required for cell-cell contact in vascular development and cardiac morphogenesis.

Mutationen im menschlichen gen Filamin A (FLNA) st&ouml;ren neuronale Migration in der Hirnrinde und Herz-Kreislauf-Defekte verursachen. Vollst&auml;ndiger Verlust der Flna bei M&auml;usen f&uuml;hrt zu embryonalen T&ouml;dlichkeit mit schweren strukturellen Herzfehler, die Ventrikel, Atrien, sowie Abfluss Traktate und weit verbreitete aberrant Musterung vaskul&auml;re. Trotz dieser weit verbreiteten Entwicklungsst&ouml;rungen M&auml;ngel erscheint Migration und Motilit&auml;t von vielen Zelltypen nicht betroffen sein. Stattdessen zeigen Flna-Null Embryonen St&ouml;rung epitheliale und endothelial Organisation und aberrante Adherens Junctions Blutgef&auml;&szlig;e, Herz, Gehirn und anderen Geweben zu entwickeln. Wesentliche Rollen f&uuml;r FLNA im interzellul&auml;ren Verbindungen bieten einen Mechanismus f&uuml;r diverse Entwicklungsst&ouml;rungen M&auml;ngel bei Patienten mit FLNA Mutationen gesehen.

Filamin A (FLNA) ist erforderlich f&uuml;r Zell-Zell-Kontakt in vaskul&auml;re Entwicklung und kardiale Morphogenese.

The actin cytoskeleton is critical for tip growth in plants. Profilin is the main monomer actin binding protein in plant cells. The moss Physcomitrella patens has three profilin genes, which are monophyletic, suggesting a single ancestor for plant profilins. Here, we used RNA interference (RNAi) to determine the loss-of-function phenotype of profilin. Reduction of profilin leads to a complete loss of tip growth and a partial inhibition of cell division, resulting in plants with small rounded cells and fewer cells. We silenced all profilins by targeting their 3' untranslated region sequences, enabling complementation analyses by expression of profilin coding sequences. We show that any moss or a lily (Lilium longiflorum) profilin support tip growth. Profilin with a mutation in its actin binding site is unable to rescue profilin RNAi, while a mutation in the poly-l-proline binding site weakly rescues. We show that moss tip growing cells contain a prominent subapical cortical F-actin structure composed of parallel actin cables. Cells lacking profilin lose this structure; instead, their F-actin is disorganized and forms polarized cortical patches. Plants expressing the actin and poly-l-proline binding mutants exhibited similar F-actin disorganization. These results demonstrate that profilin and its binding to actin are essential for tip growth. Additionally, profilin is not needed for formation of F-actin, but profilin and its interactions with actin and poly-l-proline ligands are required to properly organize F-actin.

Profilin is essential for tip growth in the moss Physcomitrella patens.

Das Aktin-Zytoskelett ist kritisch f&uuml;r Tipp Wachstum der Pflanzen. Profilin ist die wichtigste Monomer Aktin verbindliches Protein in Pflanzenzellen. Das Moos Physcomitrella Patens hat drei profilin Gene, die monophyletische, was auf einen einzigen Vorfahren f&uuml;r Pflanzen Profilins sind. Hier haben wir RNA-Interferenz (RNAi) verwendet, um zu bestimmen, der Verlust der Funktion-Ph&auml;notyp von profilin. Reduzierung von profilin f&uuml;hrt zu einem v&ouml;lligen Verlust von Tipp-Wachstum und einen partiellen Hemmung der Zellteilung, was zu Pflanzen mit kleinen abgerundeten Zellen und weniger Zellen. Wir zum Schweigen gebracht alle Profilins durch ihre 3&#39; nicht &uuml;bersetzte Region Sequenzen, gezielte Erg&auml;nzung Analysen durch Ausdruck von profilin Codierung Sequenzen aktivieren. Wir zeigen, dass jeder Moos oder eine Lilie (Lilie) profilin Unterst&uuml;tzung Tipp Wachstum. Profilin mit einer Mutation in den Aktin-Bindungsstelle kann nicht retten, profilin RNAi, w&auml;hrend eine Mutation in der Poly-l-Prolin Bindung Website schwach rettet. Wir zeigen, dass Moos Tipp wachsenden Zellen eine prominente retroflexer kortikale F-Actin Struktur setzt sich aus parallelen Aktin-Kabel enthalten. Zellen fehlt profilin verlieren diese Struktur; stattdessen ihre F-Actin ist unorganisiert und polarisierte kortikale Flecken bildet. Pflanzen, die Ausdruck der Aktin und Poly-l-Prolin Bindung Mutanten ausgestellt &auml;hnlich F-Actin Desorganisation. Diese Ergebnisse zeigen, dass profilin und seine Bindung an Aktin f&uuml;r Tipp-Wachstum notwendig sind. Dar&uuml;ber hinaus profilin ist nicht erforderlich f&uuml;r die Bildung von F-Actin, aber profilin und seine Interaktionen mit Aktin und Poly-l-Prolin-Liganden sind erforderlich, F-Actin richtig zu organisieren.

Profilin ist essentiell f&uuml;r Tipp Wachstum in das Moos Physcomitrella Patens.

We characterized the role of protein tyrosine phosphatase (PTP)-alpha in focal adhesion (FA) formation and remodeling using wild-type and PTPalpha-deficient (PTPalpha(-/-)) cells. Compared with wild-type cells, spreading PTPalpha(-/-) fibroblasts displayed fewer leading edges and formed elongated alpha-actinin-enriched FA at the cell periphery. These features suggest the presence of slowly remodeling cell adhesions and were phenocopied in human fibroblasts in which PTPalpha was knocked down using short interfering RNA (siRNA) or in NIH-3T3 fibroblasts expressing catalytically inactive (C433S/C723S) PTPalpha. Fluorescence recovery after photobleaching showed slower green fluorescence protein-alpha-actinin recovery in the FA of PTPalpha(-/-) than wild-type cells. These alterations correlated with reduced cell spreading, adhesion, and polarization and retarded contraction of extracellular matrices in PTPalpha(-/-) fibroblasts. Activation of Rac1 and its recruitment to FA during spreading were diminished in cells expressing C433S/C723S PTPalpha. Rac1(-/-) cells also displayed abnormally elongated and peripherally distributed FA that failed to remodel. Conversely, expression of constitutively active Rac1 restored normal FA remodeling in PTPalpha(-/-) cells. We conclude that PTPalpha is required for remodeling of FA during cell spreading via a pathway involving Rac1.

Tyrosine phosphatase PTPalpha regulates focal adhesion remodeling through Rac1 activation.

Wir zeichnen sich die Rolle der Protein-Tyrosin-Phosphatase (PTP)-alpha in fokale Adh&auml;sion (FA) Bildung und Umbau mit Wildtyp und PTPalpha-Mangel (PTPalpha(-/-))-Zellen. Im Vergleich zu Wildtyp Zellen, Verbreitung PTPalpha(-/-) Fibroblasten angezeigt weniger Vorderkanten und l&auml;ngliche Alpha-Actinin angereicherten FA an der Peripherie der Zelle gebildet. Diese Features vorschlagen das Vorhandensein von langsam Umbau Zelle Adh&auml;sionen und waren phenocopied in menschlichen Fibroblasten, in denen PTPalpha abgerissen wurde mit kurzen interfering RNA (SiRNA) oder NIH-3T3 Fibroblasten katalytisch inaktiv (C433S/C723S) PTPalpha zum Ausdruck zu bringen. Fluoreszenz-Wiederherstellung nach Photobleaching langsamer gr&uuml;ne Fluoreszenz-Protein-Alpha-Actinin-Erholung in den FA PTPalpha(-/-) als Wildtyp Zellen zeigte. Diese Ver&auml;nderungen korreliert mit Zelle verbreiten, Adh&auml;sion und Polarisation und Kontraktion der extrazellularen Matrizen in PTPalpha(-/-) Fibroblasten gehemmt. Aktivierung der Rac1 und seine Einstellung zu FA w&auml;hrend der Ausbreitung wurden abgenommen, in Zellen, die C433S/C723S PTPalpha zum Ausdruck zu bringen. Rac1(-/-) Zellen auch ungew&ouml;hnlich angezeigt l&auml;nglich und am Rande verteilt FA, die nicht umgestalten. Umgekehrt wiederhergestellt Ausdruck der konstitutiv aktive Rac1 normalen FA Umbau in PTPalpha(-/-) Zellen. Wir schlie&szlig;en, dass PTPalpha f&uuml;r Umbau des FA w&auml;hrend Zelle verbreitet sich &uuml;ber einen Weg mit Rac1 erforderlich ist.

Tyrosin-Phosphatase PTPalpha regelt die fokale Adh&auml;sion Umbau durch Rac1-Aktivierung.

Actin depolymerizing factor (ADF)/cofilin is important for regulating actin dynamics, and in plants is thought to be required for tip growth. However, the degree to which ADF is necessary has been elusive because of the presence of multiple ADF isoforms in many plant species. In the moss Physcomitrella patens, ADF is encoded by a single, intronless gene. We used RNA interference to demonstrate that ADF is essential for plant viability. Loss of ADF dramatically alters the organization of the F-actin cytoskeleton, and leads to an inhibition of tip growth. We show that ADF is subject to phosphorylation in vivo, and using complementation studies we show that mutations of the predicted phosphorylation site partially rescue plant viability, but with differential affects on tip growth. Specifically, the unphosphorylatable ADF S6A mutant generates small polarized plants with normal F-actin organization, whereas the phosphomimetic S6D mutant generates small, unpolarized plants with a disorganized F-actin cytoskeleton. These data indicate that phosphoregulation at serine 6 is required for full ADF function in vivo, and, in particular, that the interaction between ADF and actin is important for tip growth.

Actin depolymerizing factor is essential for viability in plants, and its phosphoregulation is important for tip growth.

Aktin depolymerizing Faktor (ADF) / Cofilin ist wichtig f&uuml;r die Regulierung der Aktin-Dynamik und in Pflanzen wird gedacht, um f&uuml;r Tipp-Wachstum erforderlich. Inwieweit die ADF notwendig ist hat jedoch wegen der Anwesenheit von mehreren ADF-Isoformen in vielen Pflanzenarten tr&uuml;gerisch erwiesen. In das Moos Physcomitrella Patens, ADF ist eine einzelne, intronless-Gen codiert. Wir verwendeten RNA-Interferenz um zu demonstrieren, dass ADF f&uuml;r Pflanzen &Uuml;berleben wichtig. Verlust der ADF dramatisch ver&auml;ndert die Organisation des Zytoskeletts F-Actin und f&uuml;hrt zu einer Hemmung der Tipp Wachstum. Wir zeigen, dass ADF in-vivo Phosphorylierung unterliegt und mit Erg&auml;nzung-Studien, die wir zeigen, dass Mutationen der vorhergesagten Phosphorylierung Website teilweise Pflanze Lebensf&auml;higkeit retten, aber mit Differential Tipp Wachstum beeinflusst. Insbesondere generiert die unphosphorylatable ADF S6A Mutant kleine polarisierte Pflanzen mit normalen F-Actin-Organisation, w&auml;hrend der Phosphomimetic S6D-Mutant kleine, unpolarisiertes Pflanzen mit einer desorganisierten F-Actin-Zytoskeletts generiert. Diese Daten zeigen, dass Phosphoregulation bei Serin 6 ist erforderlich f&uuml;r volle ADF-Funktion, die in-vivo und, vor allem, dass die Interaktion zwischen ADF und Actin Tipp Wachstum wichtig ist.

Aktin depolymerizing Faktor ist wichtig f&uuml;r die Rentabilit&auml;t in Pflanzen, und seine Phosphoregulation ist wichtig f&uuml;r Tipp-Wachstum.

Rac activation by integrins is essential for cell spreading, migration, growth and survival. Based mainly on overexpression of dominant-negative mutants, RhoG has been proposed to mediate integrin-dependent Rac activation upstream of ELMO and Dock180. RhoG-knockout mice, however, display no significant developmental or functional abnormalities. To clarify the role of RhoG in integrin-mediated signaling, we developed a RhoG-specific antibody, which, together with shRNA-mediated knockdown, allowed analysis of the endogenous protein. Despite dramatic effects of dominant-negative constructs, nearly complete RhoG depletion did not substantially inhibit cell adhesion, spreading, migration or Rac activation. Additionally, RhoG was not detectably activated by adhesion to fibronectin. Using Rac1(-/-) cells, we found that constitutively active RhoG induced membrane ruffling via both Rac-dependent and -independent pathways. Additionally, endogenous RhoG was important for Rac-independent cell migration. However, RhoG did not significantly contribute to cell spreading even in these cells. These data therefore clarify the role of RhoG in integrin signaling and cell motility.

Endogenous RhoG is dispensable for integrin-mediated cell spreading but contributes to Rac-independent migration.

RAC-Aktivierung durch Integrine unbedingt Zelle verbreiten, Migration, Wachstum und &uuml;berleben. Basiert vor allem auf eine &Uuml;berexpression von Dominant Negative Mutanten, wurde RhoG vorgeschlagen, Integrin-abh&auml;ngige Rac Aktivierung stromaufw&auml;rts von ELMO und Dock180 zu vermitteln. RhoG-Knockout-M&auml;use, zeigen jedoch keine signifikanten Entwicklungsst&ouml;rungen oder funktionellen Anomalien. Die Rolle der RhoG in Integrin-vermittelte Signalisierung zu kl&auml;ren, entwickelten wir einen RhoG-spezifische Antik&ouml;rper, denen zusammen mit ShRNA-vermittelte Niederschlag, Analyse des endogenen Proteins gestattet. Trotz der dramatischen Auswirkungen der Dominant-negativen Konstrukte fast vollst&auml;ndige RhoG Ersch&ouml;pfung nicht wesentlich Zelladh&auml;sion, Verbreitung, Migration oder Rac Aktivierung hemmen. Dar&uuml;ber hinaus wurde RhoG nicht detectably durch Adh&auml;sion auf Fibronektin aktiviert. Mit Rac1(-/-) Zellen, fanden wir, dass konstitutiv aktive RhoG Membran &uuml;ber Rac-abh&auml;ngige und -unabh&auml;ngige Wege ruffling induziert. Dar&uuml;ber hinaus war die endogene RhoG wichtig f&uuml;r Rac-unabh&auml;ngige Zellmigration. RhoG jedoch nicht wesentlich zu Zelle verbreiten sogar in diesen Zellen beitragen. Diese Daten werden daher die Rolle der RhoG in Integrin-Signalisierung und Zelle Motilit&auml;t kl&auml;ren.

Endogene RhoG ist entbehrlich f&uuml;r die Verbreitung von Integrin-vermittelte Zelle, sondern tr&auml;gt zur Rac-unabh&auml;ngige Migration.

Actin is essential for tip growth in plants. However, imaging actin in live plant cells has heretofore presented challenges. In previous studies, fluorescent probes derived from actin-binding proteins often alter growth, cause actin bundling and fail to resolve actin microfilaments.

Lifeact-mEGFP reveals a dynamic apical F-actin network in tip growing plant cells.

Aktin ist essentiell f&uuml;r Tipp Wachstum der Pflanzen. Jedoch hat imaging Aktin in live Pflanzenzellen bisher vor Herausforderungen gestellt. In den vorhergehenden Studien fluoreszierende Sonden abgeleitet von Aktin-bindende Proteine oft ver&auml;ndern Wachstum, verursachen Aktin B&uuml;ndelung und Mikrofilamenten Aktin l&ouml;sen.

Lifeact-mEGFP offenbart ein dynamisches apikale F-Actin-Netzwerk in Spitze wachsenden Pflanzenzellen.

Formins are present in all eukaryotes and are essential for the creation of actin-based structures responsible for diverse cellular processes. Because multicellular organisms contain large formin gene families, establishing the physiological functions of formin isoforms has been difficult. Using RNAi, we analyzed the function of all 9 formin genes within the moss Physcomitrella patens. We show that plants lacking class II formins (For2) are severely stunted and composed of spherical cells with disrupted actin organization. In contrast, silencing of all other formins results in normal elongated cell morphology and actin organization. Consistent with a role in polarized growth, For2 are apically localized in growing cells. We show that an N-terminal phosphatase tensin (PTEN)-like domain mediates apical localization. The PTEN-like domain is followed by a conserved formin homology (FH)1-FH2 domain, known to promote actin polymerization. To determine whether apical localization of any FH1-FH2 domain mediates polarized growth, we performed domain swapping. We found that only the class II FH1-FH2, in combination with the PTEN-like domain, rescues polarized growth, because it cannot be replaced with a similar domain from a For1. We used in vitro polymerization assays to dissect the functional differences between these FH1-FH2 domains. We found that both the FH1 and the FH2 domains from For2 are required to mediate exceptionally rapid rates of actin filament elongation, much faster than any other known formin. Thus, our data demonstrate that rapid rates of actin elongation are critical for driving the formation of apical filamentous actin necessary for polarized growth.

Rapid formin-mediated actin-filament elongation is essential for polarized plant cell growth.

Formins sind in allen Eukaryoten vorhanden und f&uuml;r die Schaffung des Aktin-basierte Strukturen verantwortlich f&uuml;r diverse zellul&auml;re Prozesse unverzichtbar. Da Mehrzeller Gro&szlig;familien formin gen enthalten, war zur Gr&uuml;ndung der physiologischen Funktionen von Pau Isoformen schwierig. Verwendung von RNAi wir analysiert die Funktion alle 9 braun Gene in das Moos Physcomitrella Patens. Wir zeigen, dass Pflanzen fehlt Klasse II Formins (f&uuml;r 2) sind stark verk&uuml;mmert und bestehend aus kugelf&ouml;rmigen Zellen mit gest&ouml;rte Aktin-Organisation. Im Gegensatz zum Schweigen aller anderen Formins Ergebnisse in normalen l&auml;nglichen Zelle Morphologie und Actin-Organisation. In &Uuml;bereinstimmung mit eine Rolle f&uuml;r polarisierte Wirtschaftswachstum, f&uuml;r 2 sind apically lokalisiert in wachsende Zellen. Wir zeigen, dass eine N-terminale Phosphatase tensin (PTEN)-wie Dom&auml;ne apikale Lokalisierung vermittelt. Die PTEN-&auml;hnliche Dom&auml;ne folgt eine konservierte formin-Homologie (FH) 1-FH2 Gebiets bekannt, Aktin-Polymerisation zu f&ouml;rdern. Um festzustellen, ob die apikale Lokalisierung einer beliebigen Dom&auml;ne FH1-FH2 polarisierte Wachstum vermittelt, f&uuml;hrten wir die Dom&auml;ne zu tauschen. Wir fanden, dass nur die Klasse II FH1-FH2, in Kombination mit der PTEN-&auml;hnliche Dom&auml;ne polarisierter Wachstum, rettet, weil es nicht mit einer &auml;hnlichen Domain aus einem For1 ersetzt werden kann. Wir haben in-vitro-Polymerisation Proben, um die funktionalen Unterschiede zwischen diesen FH1-FH2-Dom&auml;nen zu zergliedern. Wir fanden, dass die FH1 und FH2 Dom&auml;nen aus f&uuml;r 2 erforderlich sind, um die au&szlig;ergew&ouml;hnlich schnelle Preise von Aktin-Filamenten Dehnung, viel schneller als alle anderen bekannten Pau zu vermitteln. So zeigen unsere Daten, dass rapid Preise der Aktin-Dehnung kritisch f&uuml;r das fahren der Bildung von apikalen filament&ouml;sen Aktin f&uuml;r polarisierte Wachstum erforderlich sind.

Schnelle Pau-vermittelte Aktin-Filament Dehnung unbedingt polarisierten Zelle Pflanzenwachstum.

Wir entwickelten eine einfache und schnelle Methode um temperaturempfindliche Allele der wesentlichen Pflanzengene zu identifizieren. Wir verwendeten prim&auml;ren und terti&auml;ren Strukturinformationen R&uuml;ckst&auml;nde im Kern des Proteins des Interesses zu identifizieren. Diese R&uuml;ckst&auml;nde wurden mutiert und f&uuml;r Temperaturempfindlichkeit, nutzen die au&szlig;ergew&ouml;hnlich schnelle 1-Woche-Complementation Assay in das Moos Physcomitrella Patens getestet. Als Test-Molek&uuml;le wir die profilin-Aktin-bindende Proteine und Aktin-depolymerizing Faktor ausgew&auml;hlt, weil sie sind essenziell und ihren Verlust der Funktion-Ph&auml;notyp k&ouml;nnen vollst&auml;ndig gerettet werden. Eine kleine Anzahl von Kandidaten Mutanten Screening, haben wir erfolgreich temperaturempfindliche Allele von profilin und Aktin-depolymerizing Faktor identifiziert. Pflanzen beherbergen diese Allele wuchs gut an die permissive Temperatur von 20 Grad C bis 25 &deg; C aber zeigte einem vollst&auml;ndigen Verlust der Funktion bei der restriktiven Temperatur von 32 Grad C. insbesondere profilin Mutation identifiziert im Moos-gen aus anderen Pflanzenarten, auch Rendern sie Temperatur empfindlich auf Profilins &uuml;bertragen werden kann. Routinem&auml;&szlig;ig temperaturempfindliche Allele der wesentlichen Pflanzeneiwei&szlig; Generierung ist ein leistungsstarkes Tool f&uuml;r die Studie der Genfunktion in Pflanzen.

Schnell-Screening f&uuml;r temperaturempfindliche Allele in Pflanzen.

We developed a simple and fast method to identify temperature-sensitive alleles of essential plant genes. We used primary and tertiary structure information to identify residues in the core of the protein of interest. These residues were mutated and tested for temperature sensitivity, taking advantage of the exceptionally rapid 1-week complementation assay in the moss Physcomitrella patens. As test molecules, we selected the actin-binding proteins profilin and actin-depolymerizing factor, because they are essential and their loss-of-function phenotype can be fully rescued. Screening a small number of candidate mutants, we successfully identified temperature-sensitive alleles of both profilin and actin-depolymerizing factor. Plants harboring these alleles grew well at the permissive temperature of 20 degrees C to 25 degrees C but showed a complete loss of function at the restrictive temperature of 32 degrees C. Notably, the profilin mutation identified in the moss gene can be transferred to profilins from other plant species, also rendering them temperature sensitive. The ability to routinely generate temperature-sensitive alleles of essential plant proteins provides a powerful tool for the study of gene function in plants.

Rapid screening for temperature-sensitive alleles in plants.

We examined exocytosis during oscillatory growth in lily (Lilium formosanum and Lilium longiflorum) and tobacco (Nicotiana tabacum) pollen tubes using three markers: (1) changes in cell wall thickness by Nomarski differential interference contrast (DIC), (2) changes in apical cell wall fluorescence in cells stained with propidium iodide (PI), and (3) changes in apical wall fluorescence in cells expressing tobacco pectin methyl esterase fused to green fluorescent protein (PME-GFP). Using PI fluorescence, we quantified oscillatory changes in the amount of wall material from both lily and tobacco pollen tubes. Measurement of wall thickness by DIC was only possible with lily due to limitations of microscope resolution. PME-GFP, a direct marker for exocytosis, only provides information in tobacco because its expression in lily causes growth inhibition and cell death. We show that exocytosis in pollen tubes oscillates and leads the increase in growth rate; the mean phase difference between exocytosis and growth is -98 degrees +/- 3 degrees in lily and -124 degrees +/- 4 degrees in tobacco. Statistical analyses reveal that the anticipatory increase in wall material predicts, to a high degree, the rate and extent of the subsequent growth surge. Exocytosis emerges as a prime candidate for the initiation and regulation of oscillatory pollen tube growth.

Exocytosis precedes and predicts the increase in growth in oscillating pollen tubes.

Exozytose w&auml;hrend Oszillatorische Zunahme der Lilie (Lilium Frauenschuh und Lilie) und Tabak (Nicotiana Tabacum) Pollen Rohre mit drei Markern untersucht: (1) &Auml;nderungen in Zelle Wandst&auml;rke von Nomarski differenzielle Interferenz-Kontrast (DIC) (2) Ver&auml;nderungen im apikalen Zellwand Fluoreszenz in Zellen befleckt mit Propidium Jodid (PI) und (3) &Auml;nderungen im apikalen Wand Fluoreszenz in Zellen auszudr&uuml;cken Tabak Pektin Methyl Esterase zu gr&uuml;n fluoreszierendes Protein (PME-GFP) verschmolzen. Mit PI-Fluoreszenz, quantifiziert wir Oszillatorische &Auml;nderungen in der H&ouml;he von Mauer-Material aus Lilie und Tabak-Pollen-Rohren. Messung der Wanddicke von DIC war nur m&ouml;glich mit Lilie aufgrund von Beschr&auml;nkungen f&uuml;r Mikroskop-Aufl&ouml;sung. PME-GFP, direkte Marker f&uuml;r Exozytose, bietet nur Informationen in Tabak, weil seinen Ausdruck im Lily Wachstum Hemmung und Zelle Tod verursacht. Wir zeigen, dass Exozytose in Pollen R&ouml;hren oszilliert und f&uuml;hrt die Erh&ouml;hung der Wachstumsrate; die mittleren Phasendifferenz zwischen Wachstum und Exozytose ist-98 Grad +/-3 Grad im Lily und-124 Grad +/-4 Grad im Tabak. Statistische Analysen zeigen, dass die vorausschauende Zunahme der Wand Material, um ein hohes Ma&szlig;, die Rate und Ausma&szlig; von den nachfolgenden Wachstumsschub prognostiziert. Exozytose entpuppt sich als ein hei&szlig;er Kandidat f&uuml;r die Einleitung und die Regulierung der oszillierende Pollen Rohr Wachstum.

Exozytose vorausgeht und prognostiziert die Steigerung des Wachstums in oszillierende Pollen Rohre.

Class XI myosins are plant specific and responsible for cytoplasmic streaming. Because of the large number of myosin XI genes in angiosperms, it has been difficult to determine their precise role, particularly with respect to tip growth. The moss Physcomitrella patens provides an ideal system to study myosin XI function. P. patens has only two myosin XI genes, and these genes encode proteins that are 94% identical to each other. To determine their role in tip growth, we used RNA interference to specifically silence each myosin XI gene using 5' untranslated region sequences. We discovered that the two myosin XI genes are functionally redundant, since silencing of either gene does not affect growth or polarity. However, simultaneous silencing of both myosin XIs results in severely stunted plants composed of small rounded cells. Although similar to the phenotype resulting from silencing of other actin-associated proteins, we show that this phenotype is not due to altered actin dynamics. Consistent with a role in tip growth, we show that a functional, full-length fusion of monomeric enhanced green fluorescent protein (mEGFP) to myosin XI accumulates at a subcortical, apical region of actively growing protonemal cells.

Myosin XI is essential for tip growth in Physcomitrella patens.

Klasse XI Myosins sind spezifische und verantwortlich f&uuml;r das streaming von zytoplasmatischen Anlage. Aufgrund der gro&szlig;en Anzahl der Myosin-XI-Gene in Angiospermen war es schwierig, ihre genaue Rolle, insbesondere im Hinblick auf die Tipp-Wachstum zu bestimmen. Das Moos Physcomitrella Patens bietet ein ideales System Myosin XI-Funktion zu studieren. P. Patens hat nur zwei Myosin XI-Gene, und diese Gene kodieren Proteine, die 94 % identisch zueinander sind. Um ihre Rolle in der Spitze Wachstum zu bestimmen, verwendet wir RNA-Interferenz, speziell Stille jedes Myosin-XI-gen mit 5&#39; nicht &uuml;bersetzte Region Sequenzen. Wir entdeckten, dass die beiden Myosin-XI-Gene funktional &uuml;berfl&uuml;ssig, sind da Unterdr&uuml;ckender entweder gen Wachstum oder Polarit&auml;t unber&uuml;hrt. F&uuml;hrt jedoch gleichzeitig zum Schweigen der beiden Myosin-XIs stark verkr&uuml;ppelte Pflanzen, bestehend aus kleinen abgerundeten Zellen. Obwohl &auml;hnlich wie der Ph&auml;notyp aus Unterdr&uuml;ckender anderer Aktin-assoziierte Proteine, zeigen wir, dass dieser Ph&auml;notyp nicht durch ver&auml;nderte Aktin-Dynamik ist. In &Uuml;bereinstimmung mit eine Rolle bei der Tipp-Wachstum, zeigen wir, dass eine funktionale, in Fusion der Monomer gr&uuml;nes fluoreszierendes Protein (mEGFP), Myosin, die XI an eine subkortikale und apikalen Region verbessert der aktiv wachsende protonemal Zellen ansammelt.

Myosin XI unbedingt Tipp Wachstum in Physcomitrella Patens.

The remodeling of actin networks is required for a variety of cellular processes in eukaryotes. In plants, several actin binding proteins have been implicated in remodeling cortical actin filaments (F-actin). However, the extent to which these proteins support F-actin dynamics in planta has not been tested. Using reverse genetics, complementation analyses, and cell biological approaches, we assessed the in vivo function of two actin turnover proteins: actin interacting protein1 (AIP1) and actin depolymerizing factor (ADF). We report that AIP1 is a single-copy gene in the moss Physcomitrella patens. AIP1 knockout plants are viable but have reduced expansion of tip-growing cells. AIP1 is diffusely cytosolic and functions in a common genetic pathway with ADF to promote tip growth. Specifically, ADF can partially compensate for loss of AIP1, and AIP1 requires ADF for function. Consistent with a role in actin remodeling, AIP1 knockout lines accumulate F-actin bundles, have fewer dynamic ends, and have reduced severing frequency. Importantly, we demonstrate that AIP1 promotes and ADF is essential for cortical F-actin dynamics.

Actin interacting protein1 and actin depolymerizing factor drive rapid actin dynamics in Physcomitrella patens.

Die Umgestaltung des Aktin-Netzwerke ist erforderlich f&uuml;r eine Vielzahl zellul&auml;rer Prozesse in den Eukaryotes. In Pflanzen haben mehrere Aktin-bindende Proteine in Umbau kortikale Aktin-Filamenten (F-Actin) verwickelt. Das Ausma&szlig; dieser Proteine auf dem F-Actin-Dynamik in Planta unterst&uuml;tzen wurde jedoch nicht getestet. Mit Reverser Komplementation Analysen und Zelle biologische Ans&auml;tze, wir beurteilen die in-vivo-Funktion zwei Aktin-Umsatz-Proteine: Aktin zusammenwirkendes protein1 (AIP1) und Actin depolymerizing Faktor (ADF). Wir berichten, dass AIP1 ein Single Copy-gen in das Moos Physcomitrella ist Patens. AIP1 ko Pflanzen lebensf&auml;hig aber haben reduzierte Ausbau der Spitze wachsenden Zellen. AIP1 ist diffus Zytosolische und Funktionen in einen gemeinsamen genetischen Weg mit ADF Tipp Wachstum zu f&ouml;rdern. Insbesondere ADF kann teilweise kompensieren, Verlust von AIP1 und AIP1 erfordert ADF f&uuml;r Funktion. In &Uuml;bereinstimmung mit eine Rolle bei der Umgestaltung von Aktin, AIP1 ko Linien F-Actin-B&uuml;ndel zu sammeln, haben weniger dynamische enden und haben ausgel&ouml;st Frequenz reduziert. Wichtig ist, zeigen wir, dass AIP1 f&ouml;rdert und ADF f&uuml;r kortikale F-Actin-Dynamik essentiell.

Article	Year
Rab2 GTPase regulates vesicle trafficking between the endoplasmic reticulum and the Golgi bodies and is important to pollen tube growth. The Plant cell\| PubMed ID: 11971147	2002
The regulation of actin organization by actin-depolymerizing factor in elongating pollen tubes. The Plant cell\| PubMed ID: 12215514	2002
Plant 115-kDa actin-filament bundling protein, P-115-ABP, is a homologue of plant villin and is widely distributed in cells. Plant & cell physiology\| PubMed ID: 14581634	2003
Profilin inhibits pollen tube growth through actin-binding, but not poly-L-proline-binding. Planta\| PubMed ID: 14712393	2004
Rac1-null mouse embryonic fibroblasts are motile and respond to platelet-derived growth factor. Molecular biology of the cell\| PubMed ID: 16525021	2006
Filamin A (FLNA) is required for cell-cell contact in vascular development and cardiac morphogenesis. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 17172441	2006
Profilin is essential for tip growth in the moss Physcomitrella patens. The Plant cell\| PubMed ID: 17981997	2007
Tyrosine phosphatase PTPalpha regulates focal adhesion remodeling through Rac1 activation. American journal of physiology. Cell physiology\| PubMed ID: 18216165	2008
Actin depolymerizing factor is essential for viability in plants, and its phosphoregulation is important for tip growth. The Plant journal : for cell and molecular biology\| PubMed ID: 18298672	2008
Endogenous RhoG is dispensable for integrin-mediated cell spreading but contributes to Rac-independent migration. Journal of cell science\| PubMed ID: 18505794	2008
Lifeact-mEGFP reveals a dynamic apical F-actin network in tip growing plant cells. PloS one\| PubMed ID: 19478943	2009
Rapid formin-mediated actin-filament elongation is essential for polarized plant cell growth. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 19633191	2009
Rapid screening for temperature-sensitive alleles in plants. Plant physiology\| PubMed ID: 19666707	2009
Exocytosis precedes and predicts the increase in growth in oscillating pollen tubes. The Plant cell\| PubMed ID: 19861555	2009
Myosin XI is essential for tip growth in Physcomitrella patens. The Plant cell\| PubMed ID: 20525854	2010
Actin interacting protein1 and actin depolymerizing factor drive rapid actin dynamics in Physcomitrella patens. The Plant cell\| PubMed ID: 22003077	2011

Luis Vidali

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