Cytoskeletal dynamics in interphase, mitosis and cytokinesis analysed through Agrobacterium-mediated transient transformation of tobacco BY-2 cells.

Transient transformation with Agrobacterium is a widespread tool allowing rapid expression analyses in plants. However, the available methods generate expression in interphase and do not allow the routine analysis of dividing cells. Here, we present a transient transformation method (termed 'TAMBY2') to enable cell biological studies in interphase and cell division. Agrobacterium-mediated transient gene expression in tobacco BY-2 was analysed by Western blotting and quantitative fluorescence microscopy. Time-lapse microscopy of cytoskeletal markers was employed to monitor cell division. Double-labelling in interphase and mitosis enabled localization studies. We found that the transient transformation efficiency was highest when BY-2/Agrobacterium co-cultivation was performed on solid medium. Transformants produced in this way divided at high frequency. We demonstrated the utility of the method by defining the behaviour of a previously uncharacterized microtubule motor, KinG, throughout the cell cycle. Our analyses demonstrated that TAMBY2 provides a flexible tool for the transient transformation of BY-2 with Agrobacterium. Fluorescence double-labelling showed that KinG localizes to microtubules and to F-actin. In interphase, KinG accumulates on microtubule lagging ends, suggesting a minus-end-directed function in vivo. Time-lapse studies of cell division showed that GFP-KinG strongly labels preprophase band and phragmoplast, but not the metaphase spindle.

An actin network is present in the cytoplasm throughout the cell cycle of carrot cells and associates with the dividing nucleus.

We have studied the F-actin network in cycling suspension culture cells of carrot (Daucus carota L.) using rhodaminyl lysine phallotoxin (RLP). In addition to conventional fixation with formaldehyde, we have used two different nonfixation methods before adding RLP: extracting cells in a stabilizing buffer; inducing transient pores in the plasma membrane with pulses of direct current (electroporation). These alternative methods for introducing RLP revealed additional features of the actin network not seen in aldehyde-fixed cells. The three-dimensional organization of this network in nonflattened cells was demonstrated by projecting stereopairs derived from through-focal series of computer-enhanced images. F-actin is present in interphase cells in four interconnected configurations: a meshwork surrounding the nucleus; thick cables in transvacuolar strands and deep in the cytoplasm; a finer network of bundles within the cortical cytoplasm; even finer filaments that run in ordered transverse array around the cell periphery. The actin network is organized differently during division but it does not disappear as do the cortical microtubules. RLP stains a central filamentous cortical band as the chromatin begins to condense (preprophase); it stains the mitotic spindle (as recently shown by Seagull et al. [Seagull, R. W., M. Falconer, and C. A. Weerdenburg, 1987, J. Cell Biol., 104:995-1004] for aldehyde fixed suspension cells) and the cytokinetic apparatus (as shown by Clayton, L., and C. W. Lloyd, 1985, Exp. Cell Res., 156:231-238). However, it is now shown that an additional network of F-actin persists in the cytoplasm throughout division associating in turn with the preprophase band, the mitotic spindle, and the cytokinetic phragmoplast.

Cloning and characterization of the unusual cyclin gene from an amphidiploid of Nicotiana glauca-Nicotiana langsdorffii hybrid.

Cyclins play an important role in the regulation of cell cycle progression in eukaryotic cells. As an aid to understanding the molecular nature of unregulated cell proliferation, a cDNA clone encoding a cyclin gene, GTcyc, was identified from genetic tumors. The clone contained 1095 bp including a 24 base poly(A) tail. GTcyc is an unusual cyclin gene, distantly related to mammalian cyclin D genes having 21-25% identity within the cyclin box. Northern blots showed that the genetic tumors express high levels of GTcyc relative to non-tumor hybrid tissues. Southern analysis suggests that GTcyc may be contained one or two families in genetic tumors.

Cellular basis of shoot apical meristem development.

Shoot apical meristems are composed of proliferating, embryonic type cells, that generate tissues and organs throughout the life of the plant. This review covers the cell biology of the higher plant shoot apical meristem (SAM). The first section describes the molecular basis of plant cell growth and division. The genetic mechanisms, that operate in meristem function and the identification of several key regulators of meristem behavior are described in the second section, and intercellular communication and coordination of cellular behavior in the third part. Finally, we discuss some recent results that indicate interaction between the cellular regulators, such as the cell cycle control genes and developmental regulators.

Aspergillus nidulans contains a single actin gene which has unique intron locations and encodes a gamma-actin.

The single actin gene from the filamentous fungus Aspergillus nidulans has been isolated and characterized. The only other organism reported to contain just one actin gene is another Ascomycete, the budding yeast Saccharomyces. The nucleotide sequence of the A. nidulans actin gene predicts a polypeptide containing the N-terminal sequence identifying the gamma-actin isotype. Until now this characteristic N terminus has only been reported to occur in vertebrate actin sequences. A monospecific anti-gamma-actin antiserum recognizes a single 42-kDa band in immunoblots of total Aspergillus protein. None of the six introns in the A. nidulans actin gene sequence aligns precisely with those found in other actin genes. One, unlike other known actin introns, is located in the 3'-untranslated region of the gene. The 5' and 3' ends of the gene have been characterized. The Aspergillus actin gene has a heterogeneous transcript size due to the presence of several different 3' termini. Of four characterized polyadenylated transcripts, only the longest contains a typical AATAAA polyadenylation signal near its 3' terminus. Using an integrative plasmid containing Aspergillus actin sequences and the pyr4 gene from Neurospora, the A. nidulans actin gene has been mapped to the first chromosome.

A glucoamylase::GFP gene fusion to study protein secretion by individual hyphae of Aspergillus niger.

Although Aspergillus niger is used as a host for heterologous protein production, yields are generally lower than those obtained for homologous proteins. Mechanisms of protein secretion and the secretory pathway in filamentous fungi are poorly characterised, although there is evidence to suggest that secretion occurs by a mechanism similar to that in other eukaryotes, but with proteins destined for secretion being directed to the hyphal tip. We report on a method using a glucoamylase: GFP gene fusion which allows us for the first time to monitor, in vivo, protein secretion in A. niger at the single hyphal level. A synthetic green fluorescent protein (sGFP(S65T)) was fused to truncated A. niger glucoamylase (GLA:499). Southern blot analysis of transformants confirmed that the gene fusion had successfully integrated into the A. niger genome. Confocal and fluorescence microscopy revealed that the GLA::GFP fusion protein is fluorescent in A. niger and appears to be directed to the hyphal tip. In young mycelia, hyphal cell wall fluorescence is apparent and immunogold labelling of GFP confirmed that GFP was partially localised within the hyphal cell wall. Using Western blotting, extracellular GLA::GFP was detected only in culture filtrates of young mycelia grown in a soya milk medium. The actin inhibitor latrunculin B was used to disrupt the secretion process, and its effects on the distribution of GLA::GFP were monitored.

Control of protein translation by phosphorylation of the mRNA 5'-cap-binding complex.

Initiation of mRNA translation is a key regulatory step in the control of gene expression. Microarray analysis indicates that total mRNA levels do not always reflect protein levels, since mRNA association with polyribosomes is necessary for protein synthesis. Phosphorylation of translation initiation factors offers a cost-effective and rapid way to adapt to physiological and environmental changes, and there is increasing evidence that many of these factors are subject to multiple regulatory phosphorylation events. The present article focuses on the nature of reversible phosphorylation and the function of the 5'-cap-binding complex in plants.

Cell division in Aspergillus.

Amenable to sophisticated genetic and molecular analysis, the simple filamentous fungus Aspergillus nidulans has provided some novel insights into the mechanisms and regulation of cell division. Mutational analysis has identified over fifty genes necessary for nuclear division, nuclear movement and cytokinesis. Molecular and cellular analysis of these mutants has led to the discovery of novel components of the cytoskeleton as well as to clarifying the role of established cytoskeletal proteins. Mutations leading to defects in the kinases (i.e. p34cdc2) and phosphatases (i.e. cdc25 and PP1), which are known to regulate mitosis in other eukaryotes, have been identified in Aspergillus. Additional, as yet novel, mitotic regulatory molecules, encoded by the nimA and bimE genes, have also been discovered in Aspergillus.

The bimG gene of Aspergillus nidulans, required for completion of anaphase, encodes a homolog of mammalian phosphoprotein phosphatase 1.

In Aspergillus nidulans, the temperature-sensitive, recessive cell cycle mutation bimG11 causes an elevated mitotic index at restrictive temperature and an inability to complete the anaphase separation of daughter nuclei. We have shown that this mutation has an abnormally high content of nuclear phosphoproteins and that the wild-type gene encodes a type 1 protein phosphatase. We conclude that dephosphorylation of a key protein(s) is required to complete mitosis.

Cell-cycle modulation of MPM-2-specific spindle pole body phosphorylation in Aspergillus nidulans.

MPM-2 is a monoclonal antibody that interacts with mitosis-specific phosphorylated proteins in many different organisms. Immunocytochemistry of tissue culture cells has shown that MPM-2 stains centrosomes, chromosomes, kinetochores, and spindles. In this paper, we demonstrate that MPM-2 staining colocalizes with the spindle pole body (SPB) of Aspergillus nidulans and that SPB staining varies during the mitotic cycle. In an unsynchronized population, about one-fourth to one-third of the cells stain with MPM-2 at the spindle plaques or SPBs. Nuclei in mitosis have two SPBs localized at the ends of the spindle, both of which stain with MPM-2. To determine when MPM-2 staining appears, we have examined the effects of temperature-sensitive cell-cycle mutations that block nuclear division in S or G2. Only a very small fraction of cells blocked in S-phase stain with MPM-2. In contrast, a large fraction of cells blocked in G2 stain brightly at the SPB. These data suggest that MPM-2 reactivity of SPBs appears in G2. Moreover, the fact that cells blocked in G2 showed MPM-2 staining but no spindles suggests that reactivity of SPBs occurs prior to mitosis but is not sufficient to trigger spindle formation. When G2-blocked cells were downshifted to permissive temperature, they generated a mitotic spindle with an SPB at each end. Both SPBs stained with MPM-2 in all of the mitotic cells.

Immunofluorescence microscopy of microtubules in intact cell lineages of the moss, Physcomitrella patens. I. Normal and CIPC-treated tip cells.

Monoclonal antibodies to yeast tubulin have been used to visualize the distribution of microtubules in the intact filamentous protonemata of the moss Physcomitrella patens. Protonemata were prepared for immunofluorescence by fixation in formaldehyde and cells were made permeable with Driselase. Extensive cell files were preserved by 'blotting' the moss onto glutaraldehyde-derivatized coverslips. Problems due to fluorescence from chloroplasts were obviated by extraction with dimethyl sulphoxide and the non-ionic detergent, Nonidet NP40. These improvements allowed us to determine that microtubules were present throughout the cell cycle in the apical dome of caulonemal tip cells, that was a pronounced association of microtubules with the nucleus, that 'astral' microtubules were associated with the mitotic spindle and during anaphase may be involved in reorientation of the spindle before an oblique cytokinesis in caulonemata and that the cytokinetic phragmoplast appeared identical to the structure described for higher plants. Microtubules appeared to converge at the very tip of apical caulonemal cells and this was studied further by treating cells with CIPC--a drug that is known to produce multiple microtubule-organizing centres--and which here produces multiple foci for microtubules at the tip. These observations emphasize the involvement of microtubules in tip growth, alignment of the cell plate and nuclear migration--processes that are fundamental to the morphogenesis of filamentous organisms.

A nimA-like protein kinase transcript is highly expressed in meristems of Antirrhinum majus.

In plants, cell proliferation occurs mostly within meristems but a significant amount also occurs at other well-defined sites during specific stages of development. We have developed molecular markers to follow the location and progress of cell division within multicellular plant organs and thereby gain some insight into how cell division might be regulated during morphogenesis. As in other eukaryotes, cell division in plants is regulated by a highly conserved set of protein kinases and phosphatases. The molecular information available on these molecules from other eukaryotes has allowed the design of strategies by which plant homologues can be isolated. In this report we describe the identification of a nimA-like gene from Antirrhinum majus and describe the pattern in which its transcript is expressed. Comparison of the pattern of AmnimA gene expression with that of genes which are expressed in a cell cycle-dependent manner suggests that this gene is expressed in actively dividing tissues but expression is not specific to any particular phase of the cell cycle nor specific to any particular tissue type.

Anti-tubulin antibodies locate the blepharoplast during spermatogenesis in the fern Platyzoma microphyllum R.Br.: a correlated immunofluorescence and electron-microscopic study.

The discovery that the monoclonal anti-tubulin antibody YOL 1/34 recognizes a microtubule organizing centre, the blepharoplast (which arises de novo during the latter stages of spermatogenesis in the fern, Platyzoma microphyllum), has enabled us to follow it and associated microtubules throughout most of its ontogeny. By correlating electron-microscopic and immunofluorescence observations, YOL 1/34 is seen to stain the blepharoplast uniformly at a time when no microtubules are present within the organelle. Later, staining becomes intense at the surface, concomitant with the re-location of cylindrical channels to the periphery of the blepharoplast. During anaphase of the ultimate division of the spermatid mother cell the blepharoplast moves to the spindle poles and sharpens the otherwise barrel-shaped mitotic apparatus. Prior to this stage the blepharoplast is, however, off-centre and at variable positions around the poles. Later still, in the differentiating spermatids, the blepharoplast is the focus for radiating cytoplasmic microtubules that abut directly onto the electron-dense organelle, penetrating the ribosome-free halo. The three main conclusions are: that tubulin in a pre-microtubular form is associated with the cylindrical channels that arise de novo within the previously amorphous blepharoplast and act as a template in basal body formation; that the late appearance of the blepharoplast as a focus for the spindle poles during the final mitosis provides strong argument against its functioning during spindle pole initiation (despite its ability to sharpen the poles at anaphase); that the blepharoplast does seem to act as a microtubule organizing centre in the mitotically quiescent spermatid.

A type 2A protein phosphatase gene from Aspergillus nidulans is involved in hyphal morphogenesis.

A PCR-based approach, using degenerate oligonucleotide primers, was used to isolate fragments of two genes encoding type 2A protein phosphatases from the filamentous fungus, Aspergillus nidulans. The complete genomic sequence of one of these genes, pphA, was isolated and characterised. The pphA gene was predicted to encode a 329-residue protein which is about 85% identical to mammalian protein phosphatase 2A. Ectopic expression of the wild-type pphA+ gene slightly inhibited growth in some transformants; but a mutant form of pphA, in which R259 was mutated to Q, led to slow growth, delayed germ tube emergence and mitotic defects at low temperature. These results indicate that the pphA+ gene plays an important role in hyphal growth.

Spindle formation and chromatin condensation in cells blocked at interphase by mutation of a negative cell cycle control gene.

In Aspergillus nidulans the temperature-sensitive cell cycle mutation bimE7 causes chromosome condensation and pre-anaphase spindle formation to occur at restrictive temperature. By constructing double mutants between bimE7 and S phase or G2 phase mutants and blocking DNA replication with hydroxyurea, we demonstrate that bimE7 can cause chromatin condensation and spindle formation in cells held in S or G2. Thus bimE7 overrides normal control systems that prevent mitosis from prematurely occurring during S or G2. We show that bimE7 is a loss of function mutation and propose that bimE normally functions to negatively control a positive mitotic inducing factor, possibly the cell cycle gene nimA.

The Arabidopsis D-type cyclins CycD2 and CycD3 both interact in vivo with the PSTAIRE cyclin-dependent kinase Cdc2a but are differentially controlled.

D-type cyclins (CycD) play key roles in linking the Arabidopsis cell cycle to extracellular and developmental signals, but little is known of their regulation at the post-transcriptional level or of their cyclin-dependent kinase (CDK) partners. Using new antisera to CycD2 and CycD3, we demonstrate that the CDK partner of these Arabidopsis cyclins is the PSTAIRE-containing CDK Cdc2a. Previous analysis has shown that transcript levels of CycD2 and CycD3 are regulated in response to sucrose levels and that both their mRNA levels and kinase activity are induced with different kinetics during the G(1) phase of cells reentering the division cycle from quiescence. Here we analyze the protein levels and kinase activity of CycD2 and CycD3. We show that CycD3 protein and kinase activity parallel the abundance of its mRNA and that CycD3 protein is rapidly lost from cells in stationary phase or following sucrose removal. In contrast to both CycD3 and the regulation of its own mRNA levels, CycD2 protein is present at constant levels. CycD2 kinase activity is regulated by sequestration of CycD2 protein in a form inaccessible to immunoprecipitation and probably not complexed to Cdc2a.

Plant-adapted green fluorescent protein is a versatile vital reporter for gene expression, protein localization and mitosis in the filamentous fungus, Aspergillus nidulans.

Green fluorescent protein (GFP) is a useful reporter to follow the in vivo behaviour of proteins, but the wild-type gfp gene does not function in many organisms, including many plants and filamentous fungi. We show that codon-modified forms of gfp, produced for use in plants, function effectively in Aspergillus nidulans both as gene expression reporters and as vital reporters for protein location. To demonstrate the use of these modified gfps as reporter genes we have used fluorescence to follow ethanol-induced GFP expression from the alcA promoter. Translational fusions with the modified gfp were used to follow protein location in living cells; plant ER-retention signals targeted GFP to the endoplasmic reticulum, whereas fusion to the GAL4 DNA-binding domain targeted it to the nucleus. Nuclear-targeted GFP allowed real-time observation of nuclear movement and division. These modified gfp genes should provide useful markers to follow gene expression, organelle behaviour and protein trafficking in real time.

Identification and localisation of a nucleoporin-like protein component of the plant nuclear matrix.

Salt-detergent extraction of purified plant nuclei yields a fraction enriched in putative structural proteins known as the "nuclear matrix". Compared with mammalian nuclear matrices, which contain three major proteins, plant nuclear matrices are complex, containing at least 100 polypeptides. In order to characterise more fully the plant nuclear matrix we have used antibodies raised against both yeast (Saccharomyces cerevisiae) and mammalian (rat) nuclear pore proteins. We have shown that the nuclear matrix of carrot (Daucus carota L.) contains at least one nucleoporin-like protein of about 100 kDa which is immunologically related to both the yeast nuclear pore protein NSP1 and mammalian nucleoporins (p62). Antibody labelling of a variety of plant cells at the light-microscope and electron-microscope levels confirms that this antigen is located at the nuclear pores. This, to our knowledge, is the first identification of a nuclear pore protein in plants.

Patterns of cell division revealed by transcriptional regulation of genes during the cell cycle in plants.

Transcripts from five cell cycle related genes accumulate in isolated cells dispersed throughout the actively dividing regions of plant meristems. We propose that this pattern reflects gene expression during particular phases of the cell division cycle. The high proportion of isolated cells suggests that synchrony between daughter cells is rapidly lost following mitosis. This is the first time that such a cell specific expression pattern has been described in a higher organism. Counterstaining with a DNA specific dye revealed that transcripts from three genes (two mitotic cyclins and a cdc2-like gene) accumulate during part of interphase and early mitosis whereas transcripts from a histone H4 gene are preferentially detected only in interphase cells. Double labelling for cyclin and histone H4 transcripts confirms that these genes are expressed in different cells, and therefore at different phases of the cell cycle. The results suggest that transcriptional regulation of cell cycle related genes may be important in controlling cell division in plants, and that these genes are useful markers for identifying cells at specific phases of the cell cycle within plant meristems.

Two alpha-tubulin genes of Aspergillus nidulans encode divergent proteins.

We have isolated and analyzed the tubA and tubB alpha-tubulin genes of Aspergillus nidulans. The nucleotide sequences of these genes predict polypeptides of 447 amino acids for tubA and 450 for tubB. The predicted amino acids sequences exhibit 28% divergence between the two polypeptides. This is the second known case of such high divergence between alpha-tubulins within the same species. The tubB gene is unique in that it codes for an extra glycine residue between what are usually the second and third amino acids. RNA blot analysis demonstrates that the tubA and tubB transcripts are each 1.8 kb long. The level of tubA transcript remains the same throughout the cell cycle. The level of tubB transcript does not change at any particular stage in the cell cycle but increases continuously during spore germination. The tubA gene was previously mapped to linkage group eight, and we have now mapped the tubB gene to linkage group four. Gene disruption in heterokaryons suggests that the phenotypic consequences of disruption are different for the tubA and tubB genes. Molecular disruption of tubA results in a block in nuclear division whereas in tubB it gives rise to abnormal cell and nuclear morphology.