MOR1/GEM1 has an essential role in the plant-specific cytokinetic phragmoplast

MOR1 is a member of the MAP215 family of microtubule-associated proteins and is required to establish interphase arrays of cortical microtubules in plant cells. Here we show that MOR1 binds microtubules in vivo, localizing to both cortical microtubules and to areas of overlapping microtubules in the phragmoplast. Genetic complementation of the cytokinesis-defective gemini pollen 1-1 (gem1-1) mutation with MOR1 shows that MOR1 (which is synonymous with the protein GEM1) is essential in cytokinesis. Phenotypic analysis of gem1-1 and gem1-2, which contains a T-DNA insertion, confirm that MOR1/GEM1 is essential for regular patterns of cytokinesis. Both the gem1-1 and gem1-2 mutations cause the truncation of the MOR1/GEM1 protein. In addition, the carboxy-terminal domain of the protein, which is absent in both mutants, binds microtubules in vitro. Our data show that MOR1/GEM1 has an essential role in the cytokinetic phragmoplast.     

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.     

Actin organization during the cell cycle in meristematic plant cells. Actin is present in the cytokinetic phragmoplast

The distribution and organisation of F-actin during the cell cycle of meristematic root-tip cells of Allium was investigated using a rhodamine-labelled phalloidin to stain F-actin in isolated cell preparations. Such preparations could, in addition, be stained for tubulin by immunofluorescence, enabling a comparison between F-actin and microtubule distributions in the same cell. In interphase, an extensive array of actin-filament bundles was present in the cytoplasm of elongating cells, the bundles generally following the long axis of the cell and passing in close proximity to the nucleus. In contrast, the interphase microtubule array occupied the cortex of the cell and was oriented at right angles to the actin bundles. In smaller, isodiametric cells, microfilament arrays were present but less well developed. During cell division, phalloidin-specific staining was seen in the cytokinetic phragmoplast, and co-distributed with microtubules at all stages of cell plate formation; however, neither the pre-prophase band nor the mitotic spindle were stained with phalloidin. Co-distribution of F-actin and microtubules only occurs, therefore, at cytokinesis. The relationship between microfilaments and microtubules is discussed, together with the possible role of actin in the phragmoplast.     

A plant-specific DYRK kinase DYRKP coordinates cell morphology in Marchantia polymorpha

Journal of Plant Research - Dual-specificity tyrosine phosphorylation-regulated kinases (DYRKs) are activated via the auto-phosphorylation of conserved tyrosine residues in their activation loop...     

A land plant-specific multigene family in the unicellular Mesostigma argues for its close relationship to Streptophyta

The search for the unicellular relative of Streptophyta (i.e., land plants and their closest green algal relatives, the charophytes) started many years ago and remained centered around the scaly green flagellate, Mesostigma viride. To date, despite numerous studies, the phylogenetic position of Mesostigma is still debated and the nature of the unicellular ancestor of Streptophyta remains unknown. As molecular phylogenetic studies have produced conflicting results, we constructed a M. viride expressed sequence tags library and searched for sequences that are shared between M. viride and the Streptophyta (to the exclusion of the other green algal lineages--the Chlorophyta). Here, we report a multigene family that is restricted to Streptophyta and M. viride. The phylogenetic distribution of this complex character and its potential involvement in the evolution of an important land plant adaptive trait (i.e., three-dimensional tissues) argue that Mesostigma is a close unicellular relative of Streptophyta.     

A role for carnosine in cellular maintenance

The dipeptide L-carnosine has beneficial effects on cultured human fibroblasts. Physiological concentrations in standard media prolong their in vitro lifespan and strongly reduce the normal features of senescence. Late passage cells in normal medium are rejuvenated when transferred to medium containing carnosine, and become senescent when carnosine is removed. In the absence of pyruvate, carnosine is cytotoxic to neoplastic and transformed human and rodent cells. None of these effects are seen with its optical isomer, D-carnosine.     

Molecular phylogeny and evolution of the plant-specific seven-transmembrane MLO family

Homologues of barley Mlo encode the only family of seven-transmembrane (TM) proteins in plants. Their topology, subcellular localization, and sequence diversification are reminiscent of those of G-protein coupled receptors (GPCRs) from animals and fungi. We present a computational analysis of MLO family members based on 31 full-size and 3 partial sequences, which originate from several monocot species, the dicot Arabidopsis thaliana, and the moss Ceratodon purpureus. This enabled us to date the origin of the Mlo gene family back at least to the early stages of land plant evolution. The genomic organization of the corresponding genes supports a monophyletic origin of the Mlo gene family. Phylogenetic analysis revealed five clades, of which three contain both monocot and dicot members, while two indicate class-specific diversification. Analysis of the ratio of nonsynonymous-to-synonymous changes in coding sequences provided evidence for functional constraint on the evolution of the DNA sequences and purifying selection, which appears to be reduced in the first extracellular loop of 12 closely related orthologues. The 31 full-size sequences were examined for potential domain-specific intramolecular coevolution. This revealed evidence for concerted evolution of all three cytoplasmic domains with each other and the C-terminal cytoplasmic tail, suggesting interplay of all intracellular domains for MLO function.     

A plant-specific cyclin-dependent kinase is involved in the control of G2/M progression in plants

Cyclin-dependent kinases (CDKs) control the key transitions in the eukaryotic cell cycle. All the CDKs known to control G(2)/M progression in yeast and animals are distinguished by the characteristic PSTAIRE motif in their cyclin-binding domain and are closely related. Higher plants contain in addition a number of more divergent non-PSTAIRE CDKs with still obscure functions. We show that a plant-specific type of non-PSTAIRE CDKs is involved in the control of the G(2)/M progression. In synchronized tobacco BY-2 cells, the corresponding protein, accumulated in a cell cycle-regulated fashion, peaking at the G(2)/M transition. The associated histone H1 kinase activity reached a maximum in mitosis and required a yet unidentified subunit to be fully active. Down-regulation of the associated kinase activity in transgenic tobacco plants using a dominant-negative mutation delayed G(2)/M transition. These results provide the first evidence that non-PSTAIRE CDKs are involved in the control of the G(2)/M progression in plants.     

A role for cellular senescence in birth timing

Senescence contributes to the local and systemic aging of tissues and has been associated with age-related diseases. Recently, roles for this process during pregnancy have come to light, the dysregulation of which has been associated with adverse pregnancy outcomes such as preterm birth. Here, we summarize recent advances that support a role for senescence in birth timing and propose new aspects of study in this emerging field.     

A role for the FtsQLB complex in cytokinetic ring activation revealed by an ftsL allele that accelerates division

The cytokinetic apparatus of bacteria is initially formed by the polymerization of the tubulin‐like FtsZ protein into a ring structure at midcell. This so‐called Z‐ring facilitates the recruitment of many additional proteins to the division site to form the mature divisome machine. Although the assembly pathway leading to divisome formation has been well characterized, the mechanisms that trigger cell constriction remain unclear. In this report, we study a ‘forgotten’ allele of ftsL from Escherichia coli, which encodes a conserved division gene of unknown function. We discovered that this allele promotes the premature initiation of cell division. Further analysis also revealed that the mutant bypasses the requirement for the essential division proteins ZipA, FtsK and FtsN, and partially bypasses the need for FtsA. These findings suggest that rather than serving simply as a protein scaffold within the divisome, FtsL may play a more active role in the activation of the machine. Our results support a model in which FtsL, along with its partners FtsB and FtsQ, function as part of a sensing mechanism that promotes the onset of cell wall remodeling processes needed for the initiation of cell constriction once assembly of the divisome complex is deemed complete.     

The casein kinase 1 family: participation in multiple cellular processes in eukaryotes

Phosphorylation of serine, threonine and tyrosine residues by cellular protein kinases plays an important role in the regulation of various cellular processes. The serine/threonine specific casein kinase 1 and 2 protein kinase families--(CK1 and CK2)--were among the first protein kinases that had been described. In recent years our knowledge of the regulation and function of mammalian CK1 kinase family members has rapidly increased. Extracellular stimuli, the subcellular localization of CK1 isoforms, their interaction with various cellular structures and proteins, as well as autophosphorylation and proteolytic cleavage of their C-terminal regulatory domains influence CK1 kinase activity. Mammalian CK1 isoforms phosphorylate many different substrates among them key regulatory proteins involved in the control of cell differentiation, proliferation, chromosome segregation and circadian rhythms. Deregulation and/or the incidence of mutations in the coding sequence of CK1 isoforms have been linked to neurodegenerative diseases and cancer. This review will summarize our current knowledge about the function and regulation of mammalian CK1 isoforms.     

Sphingosine kinase: biochemical and cellular regulation and role in disease

Sphingolipids have emerged as molecules whose metabolism is regulated leading to generation of bioactive products including ceramide, sphingosine, and sphingosine-1-phosphate. The balance between cellular levels of these bioactive products is increasingly recognized to be critical to cell regulation; whereby, ceramide and sphingosine cause apoptosis and growth arrest phenotypes, and sphingosine-1-phosphate mediates proliferative and angiogenic responses. Sphingosine kinase is a key enzyme in modulating the levels of these lipids and is emerging as an important and regulated enzyme. This review is geared at mechanisms of regulation of sphingosine kinase and the coming to light of its role in disease.     

Tissue and cellular distribution of the extended family of protein kinase C isoenzymes

Polyclonal isoenzyme-specific antisera were developed against four calcium-independent protein kinase C (PKC) isoenzymes (delta, epsilon, epsilon', and zeta) as well as the calcium-dependent isoforms (alpha, beta I, beta II, and gamma). These antisera showed high specificities, high titers, and high binding affinities (3-370 nM) for the peptide antigens to which they were raised. Each antiserum detected a species of the predicted molecular weight by Western blot that could be blocked with the immunizing peptide. PKC was sequentially purified from rat brain, and the calcium-dependent forms were finally resolved by hydroxyapatite chromatography. Peak I reacted exclusively with antisera to PKC gamma, peak II with PKC beta I and -beta II, and peak III with PKC alpha. These same fractions, however, were devoid of immunoreactivity for the calcium-independent isoenzymes. The PKC isoenzymes demonstrated a distinctive tissue distribution when evaluated by Western blot and immunocytochemistry. PCK delta was present in brain, heart, spleen, lung, liver, ovary, pancreas, and adrenal tissues. PKC epsilon was present in brain, kidney, and pancreas, whereas PKC epsilon' was present predominantly in brain. PKC zeta was present in most tissues, particularly the lung, brain, and liver. Both PKC delta and PKC zeta showed some heterogeneity of size among the different tissues. PKC alpha was present in all organs and tissues examined. PKC beta I and -beta II were present in greatest amount in brain and spleen. Although the brain contained the most PKC gamma immunoreactivity, some immunostaining was also seen in adrenal tissue. These studies provide the first evidence of selective organ and tissue distributions of the calcium-independent PKC isoenzymes.     

Arabidopsis Fused kinase and the Kinesin‐12 subfamily constitute a signalling module required for phragmoplast expansion

The conserved Fused kinase plays vital but divergent roles in many organisms from Hedgehog signalling in Drosophila to polarization and chemotaxis in Dictyostelium. Previously we have shown that Arabidopsis Fused kinase termed TWO‐IN‐ONE (TIO) is essential for cytokinesis in both sporophytic and gametophytic cell types. Here using in vivo imaging of GFP‐tagged microtubules in dividing microspores we show that TIO is required for expansion of the phragmoplast. We identify the phragmoplast‐associated kinesins, PAKRP1/Kinesin‐12A and PAKRP1L/Kinesin‐12B, as TIO‐interacting proteins and determine TIO‐Kinesin‐12 interaction domains and their requirement in male gametophytic cytokinesis. Our results support the role of TIO as a functional protein kinase that interacts with Kinesin‐12 subfamily members mainly through the C‐terminal ARM repeat domain, but with a contribution from the N‐terminal kinase domain. The interaction of TIO with Kinesin proteins and the functional requirement of their interaction domains support the operation of a Fused kinase signalling module in phragmoplast expansion that depends upon conserved structural features in diverse Fused kinases.     

A phosphorylation-independent role for the yeast cyclin-dependent kinase activating kinase Cak1

Cdc28 is the main cyclin-dependent kinase (CDK) directing the cell cycle in the budding yeast Saccharomyces cerevisiae. Besides cyclin binding, Cdc28 requires phosphorylation by the Cak1 kinase to achieve full activity. We have previously isolated carboxy-terminal cdc28^CST mutants that are temperature sensitive and exhibit high chromosome instability. Both phenotypes are suppressed by high copy Cak1 in a manner that is independent of its catalytic activity and conversely, combination of cdc28^CST and cak1 mutations results in synthetic lethality. Altogether, these results suggest that for the Cdc28 complexes to remain stable and active, an interaction with Cak1 is needed via the carboxyl terminus of Cdc28. We report two-hybrid assay data that support this model, and results that indicate that actively growing yeast cells require an optimum Cdc28:Cak1 ratio. While Cak1 is constitutively active and expressed, dividing cells tightly regulate Cak1 protein levels to ensure presence of adequate levels of Cdc28 CDK activity.