Biomedical subjects

C J Staiger

Publications and source records attributed to C J Staiger.

14 recordsLinked to original sources

PubMed2000

Characterization of Ricinus communis phloem profilin, RcPRO1.

The mature, functional sieve tube, which forms the conduit for assimilate distribution in higher plants, is dependent upon protein import from the companion cells for maintenance of the phloem long-distance translocation system. Using antibodies raised against proteins present in the sieve-tube exudate of Ricinus communis (castor bean) seedlings, a cDNA was cloned which encoded a putative profilin, termed RcPRO1. Expression and localization studies indicated that RcPRO1 mRNA encodes a phloem profilin, with some expression occurring in epidermal, cortex, pith and xylem tissue. Purified, recombinant RcPRO1 was functionally equivalent to recombinant maize profilin ZmPRO4 in a live cell nuclear displacement assay. The apparent equilibrium dissociation constant for RcPRO1 binding to plant monomeric (G-)actin was lower than the previously characterized maize profilins. Moreover, the affinity of RcPRO1 for poly-L-proline (PLP) was significantly higher than that for recombinant maize profilins. Within the sieve-tube exudate, profilin was present in 15-fold molar excess to actin. The data suggest that actin filament formation is prevented within the assimilate stream. These results are discussed in terms of the unique physiology of the phloem.

Actins↗

PubMed2000

Maize profilin isoforms are functionally distinct.

Profilin is an actin monomer binding protein that, depending on the conditions, causes either polymerization or depolymerization of actin filaments. In plants, profilins are encoded by multigene families. In this study, an analysis of native and recombinant proteins from maize demonstrates the existence of two classes of functionally distinct profilin isoforms. Class II profilins, including native endosperm profilin and a new recombinant protein, ZmPRO5, have biochemical properties that differ from those of class I profilins. Class II profilins had higher affinity for poly-l-proline and sequestered more monomeric actin than did class I profilins. Conversely, a class I profilin inhibited hydrolysis of membrane phosphatidylinositol-4,5-bisphosphate by phospholipase C more strongly than did a class II profilin. These biochemical properties correlated with the ability of class II profilins to disrupt actin cytoplasmic architecture in live cells more rapidly than did class I profilins. The actin-sequestering activity of both maize profilin classes was found to be dependent on the concentration of free calcium. We propose a model in which profilin alters cellular concentrations of actin polymers in response to fluctuations in cytosolic calcium concentration. These results provide strong evidence that the maize profilin gene family consists of at least two classes, with distinct biochemical and live-cell properties, implying that the maize profilin isoforms perform distinct functions in the plant.

Actins↗

PubMed1999

Latrunculin B has different effects on pollen germination and tube growth.

The actin cytoskeleton is absolutely required for pollen germination and tube growth, but little is known about the regulation of actin polymer concentrations or dynamics in pollen. Here, we report that latrunculin B (LATB), a potent inhibitor of actin polymerization, had effects on pollen that were distinct from those of cytochalasin D. The equilibrium dissociation constant measured for LATB binding to maize pollen actin was determined to be 74 nM. This high affinity for pollen actin suggested that treatment of pollen with LATB would have marked effects on actin function. Indeed, LATB inhibited maize pollen germination half-maximally at 50 nM, yet it blocked pollen tube growth at one-tenth of that concentration. Low concentrations of LATB also caused partial disruption of the actin cytoskeleton in germinated maize pollen, as visualized by light microscopy and fluorescent-phalloidin staining. The amounts of filamentous actin (F-actin) in pollen were quantified by measuring phalloidin binding sites, a sensitive assay that had not been used previously for plant cells. The amount of F-actin in maize pollen increased slightly upon germination, whereas the total actin protein level did not change. LATB treatment caused a dose-dependent depolymerization of F-actin in populations of maize pollen grains and tubes. Moreover, the same concentrations of LATB caused similar depolymerization in pollen grains before germination and in pollen tubes. These data indicate that the increased sensitivity of pollen tube growth to LATB was not due to general destabilization of the actin cytoskeleton or to decreases in F-actin amounts after germination. We postulate that germination is less sensitive to LATB than tube extension because the presence of a small population of LATB-sensitive actin filaments is critical for maintenance of tip growth but not for germination of pollen, or because germination is less sensitive to partial depolymerization of the actin cytoskeleton.

Actins↗

PubMed1998

Inactivation of the mitogen-activated protein kinase Mps1 from the rice blast fungus prevents penetration of host cells but allows activation of plant defense responses.

The rice blast fungus, Magnaporthe grisea, generates enormous turgor pressure within a specialized cell called the appressorium to breach the surface of host plant cells. Here, we show that a mitogen-activated protein kinase, Mps1, is essential for appressorium penetration. Mps1 is 85% similar to yeast Slt2 mitogen-activated protein kinase and can rescue the thermosensitive growth of slt2 null mutants. The mps1-1Delta mutants of M. grisea have some phenotypes in common with slt2 mutants of yeast, including sensitivity to cell-wall-digesting enzymes, but display additional phenotypes, including reduced sporulation and fertility. Interestingly, mps1-1Delta mutants are completely nonpathogenic because of the inability of appressoria to penetrate plant cell surfaces, suggesting that penetration requires remodeling of the appressorium wall through an Mps1-dependent signaling pathway. Although mps1-1Delta mutants are unable to cause disease, they are able to trigger early plant-cell defense responses, including the accumulation of autofluorescent compounds and the rearrangement of the actin cytoskeleton. We conclude that MPS1 is essential for pathogen penetration; however, penetration is not required for induction of some plant defense responses.

Amino Acid Sequence↗

PubMed1998

Profilin is associated with the plasma membrane in microspores and pollen.

In higher plants, a large number of isoforms for the actin monomer-binding protein profilin have been identified, whereas other organisms express only few profilins. Furthermore, plant profilin isoforms are expressed in a tissue-specific manner. These observations raise questions concerning functional and locational differences between isoforms of plant profilins. In this paper, we introduce three polyclonal antisera and one monoclonal antibody developed against purified pollen profilins from Zea mays and against recombinant maize profilin. Immunoblot analyses of native profilins and four recombinant maize pollen profilin isoforms show that three of the antibodies display a preference for certain isoforms. In situ immunofluorescence of pollen of Zea mays and two developmental stages of microspores of Betula pumila indicates that all antibodies label plasma membrane-associated domains. Thus, we show that at least some profilin isoforms are located at a distinct subcellular domain within developing microspores and, less distinctly, in mature pollen. This contrasts previously reported uniform distributions throughout the cytoplasm of mature pollen and pollen tubes. The results are discussed in light of the large number of profilins co-expressed in plants and with reference to accumulating evidence for functional differences between profilin isoforms.

Animals↗

PubMed1998

A potential signaling role for profilin in pollen of Papaver rhoeas.

Regulation of pollen tube growth is known to involve alterations in intracellular calcium levels and phosphoinositide signaling, although the mechanisms involved are unclear. However, it appears likely that pollination events involve a complex interplay between signaling pathways and components of the actin cytoskeleton in pollen. In many eukaryotic cells, actin binding proteins function as stimulus-response modulators, translating signals into alterations in the cytoplasmic architecture. In this study, we examined whether profilin, which is a member of this class of signaling intermediate, might play a similar role in pollen. We have analyzed the functional properties of native profilin from pollen of Papaver rhoeas and have investigated the effects of profilin on the phosphorylation of pollen proteins in vitro by adding a slight excess of profilin to cytosolic pollen extracts. We present clear evidence that profilin interacts with soluble pollen components, resulting in dramatic alterations in the phosphorylation of several proteins. We also show, albeit in vitro, the involvement of profilin in modulating the activity of a signaling component(s) affecting protein phosphorylation. Our data, which suggest that pollen profilin can regulate actin-based cytoskeletal protein assembly and protein kinase or phosphatase activity, indicate a possible role for the involvement of profilin in signaling pathways that may regulate pollen tube growth.

Actins↗

PubMed1998

Pollen profilin function depends on interaction with proline-rich motifs.

The actin binding protein profilin has dramatic effects on actin polymerization in vitro and in living cells. Plants have large multigene families encoding profilins, and many cells or tissues can express multiple profilin isoforms. Recently, we characterized several profilin isoforms from maize pollen for their ability to alter cytoarchitecture when microinjected into living plant cells and for their association with poly-L-proline and monomeric actin from maize pollen. In this study, we characterize a new profilin isoform from maize, which has been designated ZmPRO4, that is expressed predominantly in endosperm but is also found at low levels in all tissues examined, including mature and germinated pollen. The affinity of ZmPRO4 for monomeric actin, which was measured by two independent methods, is similar to that of the three profilin isoforms previously identified in pollen. In contrast, the affinity of ZmPRO4 for poly-L-proline is nearly twofold higher than that of native pollen profilin and the other recombinant profilin isoforms. When ZmPRO4 was microinjected into plant cells, the effect on actin-dependent nuclear position was significantly more rapid than that of another pollen profilin isoform, ZmPRO1. A gain-of-function mutant (ZmPRO1-Y6F) was created and found to enhance poly-L-proline binding activity and to disrupt cytoarchitecture as effectively as ZmPRO4. In this study, we demonstrate that profilin isoforms expressed in a single cell can have different effects on actin in living cells and that the poly-L-proline binding function of profilin may have important consequences for the regulation of actin cytoskeletal dynamics in plant cells.

Actins↗

PubMed1997

Characterization of maize (Zea mays) pollen profilin function in vitro and in live cells.

Profilin is a small, 12-15 kDa, actin-binding protein that interacts with at least three different ligands. The 1:1 interaction of profilin with globular actin (G-actin) was originally thought to provide a mechanism for sequestering actin monomers in the cytoplasm. It has recently become clear that the role of profilin in the cell is more complex, perhaps due to interactions with polyphosphoinositides and proline-rich proteins, or due to the ability to lower the critical concentration for actin assembly at the fast-growing barbed end of actin filaments. Because actin-binding proteins have been shown to behave differently with heterologous sources of actin, we characterized the interaction between maize pollen profilins and plant G-actin. The equilibrium dissociation constants measured by tryptophan fluorescence quenching were similar to those of other CaATP-G-actin-profilin complexes (Kd=1.0-1.5 microM). The ability of maize profilin isoforms to bind poly-l-proline was analysed, and the Kd values for recombinant pollen and human profilins were similar when determined by two independent methods. However, the affinity of native maize pollen profilin for poly-l-proline was substantially lower than that of any of the recombinant proteins by one of these assays. The possibility of post-translational modification of profilin in the mature pollen grain is discussed. Finally, we quantified the effects of microinjection of each profilin isoform on the cytoarchitecture of Tradescantia stamen hair cells and show that the resultant disruption can be used to compare actin-binding proteins in living cells. The results are discussed in relation to a recent model of the interphase actin array in these plant cells.

Actins↗

PubMed1996

Plant profilins rescue the aberrant phenotype of profilin-deficient Dictyostelium cells.

To characterize the function of plant profilins in vivo, we expressed two pollen specific Zea mays (maize) profilin isoforms in profilin-minus Dictyostelium discoideum mutants. In maize, profilins exist as a multigene family containing 4 or more members which are highly similar to each other but substantially less similar to profilins from animals and lower eukaryotes. Previously we have shown that D. discoideum profilin-minus cells have an aberrant phenotype due to defects in cell shape, cytokinesis, and development. These defects could be rescued by introducing the pollen-specific profilins 1 or 2 from maize using a newly constructed expression vector. Expression of the heterologous profilins in Dictyostelium clones was assayed by affinity purification of the pollen profilins with poly-proline agarose and by immunoblotting with a polyclonal antiserum raised against maize pollen profilin. In contrast to the profilin-minus mutants, Dictyostelium cells expressing plant profilins showed normal cell shape, contained less F-actin, and were able to form fruiting bodies. These data provide genetic evidence that maize pollen profilins, even though they are specific for a distinct developmental stage, share functional properties with profilin from a lower eukaryote and apparently act as G-actin-sequestering proteins in this system.

Animals↗

PubMed1994

Microinjected profilin affects cytoplasmic streaming in plant cells by rapidly depolymerizing actin microfilaments.

BACKGROUND: Cytoplasmic streaming is a conspicuous feature of plant cell behaviour, in which organelles and vesicles shuttle along cytoplasmic strands that contain actin filaments. The mechanisms that regulate streaming and the formation of actin filament networks are largely unknown, but in all likelihood involve actin-binding proteins. The monomeric actin-binding protein, profilin, is a key regulator of actin-filament dynamics in animal cells and it has recently been identified in plants as a pollen allergen. We set out to determine whether plant profilin can act as a monomeric actin-binding protein and influence actin dynamics in plant cells in vivo. RESULTS: Recombinant birch-pollen profilin was purified by polyproline affinity chromatography and microinjected into Tradescantia blossfeldiana stamen hair cells. After profilin injection, a rapid and irreversible change in cellular organization and streaming was observed: within 1-3 minutes the transvacuolar cytoplasmic strands became thinner and snapped, and cytoplasmic streaming ceased. Fluorescein-labelled-phalloidin staining confirmed that this was due to depolymerization of actin filaments. To confirm that the effects observed were due to sequestration of monomeric actin, another monomeric actin-binding protein, DNase I, was injected and found to produce comparable results. CONCLUSIONS: Profilin can act as a potent regulator of actin organization in living plant cells. Its rapid effect on the integrity of cytoplasmic strands and cytoplasmic streaming supports a model in which organelle movements depend upon microfilaments that exist in dynamic equilibrium with the pool of monomeric actin.

Actin Cytoskeleton↗

PubMed1993

The profilin multigene family of maize: differential expression of three isoforms.

Profilin is a small (12-15 kDa) actin- and phospholipid-binding protein previously known only from studies on animals and lower eukaryotes but recently identified as a birch pollen allergen. Here we have identified and characterized three members of the profilin multigene family from the plant Zea mays. Two cDNAs isolated from a maize pollen library (ZmPRO 1 and ZmPRO 3) each have a single, large open reading frame encoding a putative polypeptide 131 amino acids long with a predicted molecular weight of approximately 14 kDa. A third maize pollen cDNA (ZmPRO 2) has two in-frame translation initiation codons. Use of the first ATG would result in a polypeptide 137 amino acids long with a molecular weight of 14.8 kDa. The three maize profilins are highly homologous to each other (> 90% nucleotide and amino acid sequence identity) as well as other plant profilins but show far less similarity (30-40% amino acid sequence identity) to animal and lower eukaryote profilins. Multiple sequence alignments indicate that only nine residues are shared by all eukaryotic profilins examined. However, limited comparisons reveal domains in the NH2 and COOH termini that have a high degree of similarity suggesting functional conservation. The maize gene family size is estimated to contain three to six members based on Southern blot experiments with gene-specific and coding region probes. Northern blot analysis demonstrates that the three maize profilin cDNAs characterized here are utilized in a tissue-specific manner and are anther or pollen specific.

Actins↗

PubMed1992

Ameiotic, a gene that controls meiotic chromosome and cytoskeletal behavior in maize.

Microtubule organization during the novel cell division of ameiotic microsporocytes was examined using indirect immunofluorescence microscopy. A recessive mutation of the maize gene Ameiotic causes the replacement of meiosis I with a synchronized mitotic division (Palmer, R. G. (1971). Chromosoma 35, 233-246). All identifiable cytological features of this division, including chromosome behavior and microtubule organization, were typical of somatic cell division. Significantly, a cortical microtubule band was observed during prophase in ameiotic cells. In most somatic plant cells, a preprophase band of microtubules (PPB) predicts the cortical site where the future cell plate will join the sidewall. Similar structures, however, are absent in all meiotic and postmeiotic reproductive cells examined to date. These disruptions are consistent with a model where the wild-type Ameiotic gene encodes a product which acts during or before G2 and is necessary for initiating several independent meiotic processes, including both meiotic chromosome behavior and microtubule organization. The ameiotic mutation provides additional evidence that aspects of cytoskeletal organization unique to meiosis are genetically controlled. Finally, the presence of a PPB during the ameiotic division supports a model whereby multiple mechanisms are used to determine and maintain division plane polarity during normal meiosis.

Cell Division↗

PubMed1991

The plant cytoskeleton.

Significant progress has been made in four areas: in appreciating the speed with which cortical microtubules reorient in response to environmental signals; in a consolidated understanding of the cytoskeletal nature of the phragmosome--the device that predicts and structures the division plane in vacuolated cells; in the description of new cytoskeletal proteins; and in reports that herald an attack on the cell cycle control of cytoskeletal organization.

Cell Cycle↗

PubMed1990

Microtubule distribution in dv, a maize meiotic mutant defective in the prophase to metaphase transition.

Microsporogenesis in Zea mays, the meiotic reduction of diploid sporocytes to haploid microspores, proceeds through a well-defined developmental sequence. The ability to generate mutants that affect the process makes this an ideal system for elucidating the role of the cytoskeleton during plant development. We have used immunofluorescence microscopy to compare microtubule distribution in wild-type and mutant microsporocytes. During normal meiosis the distribution of microtubules follows a specific temporal and spatial pattern that reflects the polar nature of microspore formation. Perinuclear microtubule staining increases and the nucleus elongates in the future spindle axis during late prophase I. Metaphase I spindles with highly focused poles align along the long axis of the anther locule. Cytokinesis occurs perpendicular to the spindle axis. The second division axis shifts 90 degrees with respect to the first division plane, thereby yielding an isobilateral tetrad of microspores. Microtubule distribution patterns during meiosis suggest that a nuclear envelope-associated microtubule organizing center (MTOC) controls the organization of cytoplasmic microtubules and contributes to spindle formation. The meiotic mutant dv is defective in the transition from a prophase microtubule array to a metaphase spindle. Instead of converging to form focused poles, the metaphase spindle poles remain diffuse as in prometaphase. This defect correlates with several abnormalities in subsequent developmental events including the formation of multinucleate daughter cells, multiple microspindles during meiosis II, multiple phragmoplasts, polyads of microspores, and cytoplasmic microtubule foci. These results suggest that dv is a mutation that affects MTOC organization.

Cell Division↗