Dual localized kinesin‐12 POK2 plays multiple roles during cell division and interacts with MAP65‐3

Kinesins are versatile nano‐machines that utilize variable non‐motor domains to tune specific motor microtubule encounters. During plant cytokinesis, the kinesin‐12 orthologs, PHRAGMOPLAST ORIENTING KINESIN (POK)1 and POK2, are essential for rapid centrifugal expansion of the cytokinetic apparatus, the phragmoplast, toward a pre‐selected cell plate fusion site at the cell cortex. Here, we report on the spatio‐temporal localization pattern of POK2, mediated by distinct protein domains. Functional dissection of POK2 domains revealed the association of POK2 with the site of the future cell division plane and with the phragmoplast during cytokinesis. Accumulation of POK2 at the phragmoplast midzone depends on its functional POK2 motor domain and is fine‐tuned by its carboxy‐terminal region that also directs POK2 to the division site. Furthermore, POK2 likely stabilizes the phragmoplast midzone via interaction with the conserved microtubule‐associated protein MAP65‐3/PLEIADE, a well‐established microtubule cross‐linker. Collectively, our results suggest that dual localized POK2 plays multiple roles during plant cell division.     

Mechanisms of the Ase1/PRC1/MAP65 family in central spindle assembly

During cytokinesis, the organization of the spindle midzone and chromosome segregation is controlled by the central spindle, a microtubule cytoskeleton containing kinesin motors and non‐motor microtubule‐associated proteins. The anaphase spindle elongation 1/protein regulator of cytokinesis 1/microtubule associated protein 65 (Ase1/PRC1/MAP65) family of microtubule‐bundling proteins are key regulators of central spindle assembly, mediating microtubule crosslinking and spindle elongation in the midzone. Ase1/PRC1/MAP65 serves as a complex regulatory platform for the recruitment of other midzone proteins at the spindle midzone. Herein, we summarize recent advances in understanding of the structural domains and molecular kinetics of the Ase1/PRC1/MAP65 family. We summarize the regulatory network involved in post‐translational modifications of Ase1/PRC1 by cyclin‐dependent kinase 1 (Cdk1), cell division cycle 14 (Cdc14) and Polo‐like kinase 1 (Plk1) and also highlight multiple functions of Ase1/PRC1 in central spindle organization, spindle elongation and cytokinesis during cell division.     

Interactions between Transport Protein Particle (TRAPP) complexes and Rab GTPases in Arabidopsis

Transport Protein Particle II (TRAPPII) is essential for exocytosis, endocytosis, protein sorting and cytokinesis. In spite of a considerable understanding of its biological role, little information is known about Arabidopsis TRAPPII complex topology and molecular function. In this study, independent proteomic approaches initiated with TRAPP components or Rab‐A GTPase variants converge on the TRAPPII complex. We show that the Arabidopsis genome encodes the full complement of 13 TRAPPC subunits, including four previously unidentified components. A dimerization model is proposed to account for binary interactions between TRAPPII subunits. Preferential binding to dominant negative (GDP‐bound) versus wild‐type or constitutively active (GTP‐bound) RAB‐A2a variants discriminates between TRAPPII and TRAPPIII subunits and shows that Arabidopsis complexes differ from yeast but resemble metazoan TRAPP complexes. Analyzes of Rab‐A mutant variants in trappii backgrounds provide genetic evidence that TRAPPII functions upstream of RAB‐A2a, allowing us to propose that TRAPPII is likely to behave as a guanine nucleotide exchange factor (GEF) for the RAB‐A2a GTPase. GEFs catalyze exchange of GDP for GTP; the GTP‐bound, activated, Rab then recruits a diverse local network of Rab effectors to specify membrane identity in subsequent vesicle fusion events. Understanding GEF?Rab interactions will be crucial to unravel the co‐ordination of plant membrane traffic.     

High lipid order of Arabidopsis cell‐plate membranes mediated by sterol and DYNAMIN‐RELATED PROTEIN1A function

Membranes of eukaryotic cells contain high lipid‐order sterol‐rich domains that are thought to mediate temporal and spatial organization of cellular processes. Sterols are crucial for execution of cytokinesis, the last stage of cell division, in diverse eukaryotes. The cell plate of higher‐plant cells is the membrane structure that separates daughter cells during somatic cytokinesis. Cell‐plate formation in Arabidopsis relies on sterol‐ and DYNAMIN‐RELATED PROTEIN1A (DRP1A)‐dependent endocytosis. However, functional relationships between lipid membrane order or lipid packing and endocytic machinery components during eukaryotic cytokinesis have not been elucidated. Using ratiometric live imaging of lipid order‐sensitive fluorescent probes, we show that the cell plate of Arabidopsis thaliana represents a dynamic, high lipid‐order membrane domain. The cell‐plate lipid order was found to be sensitive to pharmacological and genetic alterations of sterol composition. Sterols co‐localize with DRP1A at the cell plate, and DRP1A accumulates in detergent‐resistant membrane fractions. Modifications of sterol concentration or composition reduce cell‐plate membrane order and affect DRP1A localization. Strikingly, DRP1A function itself is essential for high lipid order at the cell plate. Our findings provide evidence that the cell plate represents a high lipid‐order domain, and pave the way to explore potential feedback between lipid order and function of dynamin‐related proteins during cytokinesis.     

The microtubule‐associated kinase‐like protein RUNKEL functions in somatic and syncytial cytokinesis

The microtubule (MT)‐associated putative kinase RUNKEL (RUK) is an important component of the phragmoplast machinery involved in cell plate formation in Arabidopsis somatic cytokinesis. Since loss‐of‐function ruk mutants display seedling lethality, it was previously not known whether RUK functions in mature sporophytes or during gametophyte development. In this study we utilized RUK proteins that lack the N‐terminal kinase domain to further examine biological processes related to RUK function. Truncated RUK proteins when expressed in wild‐type Arabidopsis plants cause cellularization defects not only in seedlings and adult tissues but also during male meiocyte development, resulting in abnormal pollen and reduced fertility. Ultrastructural analysis of male tetrads revealed irregular and incomplete or absent intersporal cell walls, caused by disorganized radial MT arrays. Moreover, in ruk mutants endosperm cellularization defects were also caused by disorganized radial MT arrays. Intriguingly, in seedlings expressing truncated RUK proteins, the kinesin HINKEL, which is required for the activation of a mitogen‐activated protein kinase signaling pathway regulating phragmoplast expansion, was mislocalized. Together, these observations support a common role for RUK in both phragmoplast‐based cytokinesis in somatic cells and syncytial cytokinesis in reproductive cells.     

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.     

Cytokinesis in bloodstream stage Trypanosoma brucei requires a family of katanins and spastin

Microtubule severing enzymes regulate microtubule dynamics in a wide range of organisms and are implicated in important cell cycle processes such as mitotic spindle assembly and disassembly, chromosome movement and cytokinesis. Here we explore the function of several microtubule severing enzyme homologues, the katanins (KAT80, KAT60a, KAT60b and KAT60c), spastin (SPA) and fidgetin (FID) in the bloodstream stage of the African trypanosome parasite, Trypanosoma brucei. The trypanosome cytoskeleton is microtubule based and remains assembled throughout the cell cycle, necessitating its remodelling during cytokinesis. Using RNA interference to deplete individual proteins, we show that the trypanosome katanin and spastin homologues are non-redundant and essential for bloodstream form proliferation. Further, cell cycle analysis revealed that these proteins play essential but discrete roles in cytokinesis. The KAT60 proteins each appear to be important during the early stages of cytokinesis, while downregulation of KAT80 specifically inhibited furrow ingression and SPA depletion prevented completion of abscission. In contrast, RNA interference of FID did not result in any discernible effects. We propose that the stable microtubule cytoskeleton of T. brucei necessitates the coordinated action of a family of katanins and spastin to bring about the cytoskeletal remodelling necessary to complete cell division.     

Isolation and partial characterization of conditional cell division cycle mutants inChlamydomonas

Summary We have isolated a number of temperature conditional cell division cycle mutants of the unicellular plantChlamydomonas reinhardtii that are defective in single nuclear genes. Cells grow and divide normally at the permissive temperature (21 °C), but arrest in division at the restrictive temperature (33 °C). We have characterized these mutants using DNA probes and immunofluorescence techniques to localize cytoskeletal and microtubule organizing centre proteins. We describe here 3 broad classes of cell cycle mutation which result in cell cycle arrest with: unreplicated DNA (G1 arrest), duplicated DNA (G2 arrest) and multiple nuclei due to defective cytokinesis (cytokinesis arrest). The continuation of nuclear division in mutants blocked in cytokinesis provides support of an earlier hypothesis that stage specific events in theChlamydomonas cell cycle are arranged in separate dependent sequences. The mutants isolated in the present study provide insights into the role of cytoskeletal proteins in the coordination of plant cell division and the means to investigate the molecular mechanisms whereby division by multiple fission is controlled in the unicellular plantChlamydomonas.Abbreviations BB basal bodies - EMS ethylmethane sulphonate - MT microtubule - MTOC Microtubule organizing centre - NBBC nucleus-basal body connector - PAR photosynthetically active radiation     

Analysis of gemini pollen 3 mutant suggests a broad function of AUGMIN in microtubule organization during sexual reproduction in Arabidopsis

In flowering plants, male gametes arise via meiosis of diploid pollen mother cells followed by two rounds of mitotic division. Haploid microspores undergo polar nuclear migration and asymmetric division at pollen mitosis I to segregate the male germline, followed by division of the germ cell to generate a pair of sperm cells. We previously reported two gemini pollen (gem) mutants that produced twin‐celled pollen arising from polarity and cytokinesis defects at pollen mitosis I in Arabidopsis. Here, we report an independent mutant, gem3, with a similar division phenotype and severe genetic transmission defects through pollen. Cytological analyses revealed that gem3 disrupts cell division during male meiosis, at pollen mitosis I and during female gametophyte development. We show that gem3 is a hypomorphic allele (aug6‐1) of AUGMIN subunit 6, encoding a conserved component in the augmin complex, which mediates microtubule (MT)‐dependent MT nucleation in acentrosomal cells. We show that MT arrays are disturbed in gem3/aug6‐1 during male meiosis and pollen mitosis I using fluorescent MT‐markers. Our results demonstrate a broad role for the augmin complex in MT organization during sexual reproduction, and highlight gem3/aug6‐1 mutants as a valuable tool for the investigation of augmin‐dependent MT nucleation and dynamics in plant cells.     

Localization and evolution of septins in algae

Septins are a group of GTP‐binding proteins that are multi‐functional, with a well‐known role in cytokinesis in animals and fungi. Although the functions of septins have been thoroughly studied in opisthokonts (fungi and animals), the function and evolution of plant/algal septins are not as well characterized. Here we describe septin localization and expression in the green algae Nannochloris bacillaris and Marvania geminata. The present data suggest that septins localize at the division site when cytokinesis occurs. In addition, we show that septin homologs may be found only in green algae, but not in other major plant lineages, such as land plants, red algae and glaucophytes. We also found other septin homolog‐possessing organisms among the diatoms, Rhizaria and cryptomonad/haptophyte lineages. Our study reveals the potential role of algal septins in cytokinesis and/or cell elongation, and confirms that septin genes appear to have been lost in the Plantae lineage, except in some green algae.     

Mechanisms of kinesin‐7 CENP‐E in kinetochore–microtubule capture and chromosome alignment during cell division

Chromosome congression is essential for faithful chromosome segregation and genomic stability in cell division. Centromere‐associated protein E (CENP‐E), a plus‐end‐directed kinesin motor, is required for congression of pole‐proximal chromosomes in metaphase. CENP‐E accumulates at the outer plate of kinetochores and mediates the kinetochore‐microtubule capture. CENP‐E also transports the chromosomes along spindle microtubules towards the equatorial plate. CENP‐E interacts with Bub1‐related kinase, Aurora B and core kinetochore components during kinetochore–microtubule attachment. In this review, we introduce the structures and mechanochemistry of kinesin‐7 CENP‐E. We highlight the complicated interactions between CENP‐E and partner proteins during chromosome congression. We summarise the detailed roles and mechanisms of CENP‐E in mitosis and meiosis, including the kinetochore–microtubule capture, chromosome congression/alignment in metaphase and the regulation of spindle assembly checkpoint. We also shed a light on the roles of CENP‐E in tumourigenesis and CENP‐E's specific inhibitors.     

A specific role for Arabidopsis TRAPPII in post-Golgi trafficking that is crucial for cytokinesis and cell polarity

Cytokinesis and cell polarity are supported by membrane trafficking from the trans-Golgi network (TGN), but the molecular mechanisms that promote membrane trafficking from the TGN are poorly defined in plant cells. Here we show that TRAPPII in Arabidopsis regulates the post-Golgi trafficking that is crucial for assembly of the cell plate and cell polarity. Disruptions of AtTRS120 or AtTRS130, two genes encoding two key subunits of TRAPPII, result in defective cytokinesis and cell polarity in embryogenesis and seedling development. In attrs120 and attrs130, the organization and trafficking in the endoplasmic reticulum (ER)-Golgi interface are normal. However, post-Golgi trafficking to the cell plate and to the cell wall, but not to the vacuole, is impaired. Furthermore, TRAPPII is required for the selective transport of PIN2, but not PIN1, to the plasma membrane. We revealed that AtTRS130 is co-localized with RAB-A1c. Expression of constitutively active RAB-A1c partially rescues attrs130. RAB-A1c, which resides at the TGN, is delocalized to the cytosol in attrs130. We propose that TRAPPII in Arabidopsis acts upstream of Rab-A GTPases in post-Golgi membrane trafficking in plant cells.     

Cycling in a crowd: Coordination of plant cell division,growth, and cell fate

The reiterative organogenesis that drives plant growth relies on the constant production of new cells, which remain encased by interconnected cell walls. For these reasons, plant morphogenesis strictly depends on the rate and orientation of both cell division and cell growth. Important progress has been made in recent years in understanding how cell cycle progression and the orientation of cell divisions are coordinated with cell and organ growth and with the acquisition of specialized cell fates. We review basic concepts and players in plant cell cycle and division, and then focus on their links to growth-related cues, such as metabolic state, cell size, cell geometry, and cell mechanics, and on how cell cycle progression and cell division are linked to specific cell fates. The retinoblastoma pathway has emerged as a major player in the coordination of the cell cycle with both growth and cell identity, while microtubule dynamics are central in the coordination of oriented cell divisions. Future challenges include clarifying feedbacks between growth and cell cycle progression, revealing the molecular basis of cell division orientation in response to mechanical and chemical signals, and probing the links between cell fate changes and chromatin dynamics during the cell cycle.

Plant cell cycle and division are linked to specific cell fates and respond to growth-related cues, such as metabolic state, cell size, cell shape, and mechanical stress.     

Regulation of cytoskeleton‐associated protein activities: Linking cellular signals to plant cytoskeletal function

The plant cytoskeleton undergoes dynamic remodeling in response to diverse developmental and environmental cues. Remodeling of the cytoskeleton coordinates growth in plant cells, including trafficking and exocytosis of membrane and wall components during cell expansion, and regulation of hypocotyl elongation in response to light. Cytoskeletal remodeling also has key functions in disease resistance and abiotic stress responses. Many stimuli result in altered activity of cytoskeleton-associatedproteins,microtubuleassociated proteins(MAPs) and actin-binding proteins(ABPs). MAPs and ABPs are the main players determining the spatiotemporally dynamic nature of the cytoskeleton, functioning in a sensory hub that decodes signals to modulate plant cytoskeletal behavior. Moreover, MAP and ABP activities and levels are precisely regulated during development and environmental responses, but our understanding of this process remains limited. In this review, we summarize the evidence linking multiple signaling pathways, MAP and ABP activities and levels, and cytoskeletal rearrangements in plant cells. We highlight advances in elucidating the multiple mechanisms that regulate MAP and ABP activities and levels, including calcium and calmodulin signaling, ROP GTPase activity, phospholipid signaling, and post-translational modifications.     

Microtubule networks for plant cell division

During cytokinesis the cytoplasm of a cell is divided to form two daughter cells. In animal cells, the existing plasma membrane is first constricted and then abscised to generate two individual plasma membranes. Plant cells on the other hand divide by forming an interior dividing wall, the so-called cell plate, which is constructed by localized deposition of membrane and cell wall material. Construction starts in the centre of the cell at the locus of the mitotic spindle and continues radially towards the existing plasma membrane. Finally the membrane of the cell plate and plasma membrane fuse to form two individual plasma membranes. Two microtubule-based cytoskeletal networks, the phragmoplast and the pre-prophase band (PPB), jointly control cytokinesis in plants. The bipolar microtubule array of the phragmoplast regulates cell plate deposition towards a cortical position that is templated by the ring-shaped microtubule array of the PPB. In contrast to most animal cells, plants do not use centrosomes as foci of microtubule growth initiation. Instead, plant microtubule networks are striking examples of self-organizing systems that emerge from physically constrained interactions of dispersed microtubules. Here we will discuss how microtubule-based activities including growth, shrinkage, severing, sliding, nucleation and bundling interrelate to jointly generate the required ordered structures. Evidence mounts that adapter proteins sense the local geometry of microtubules to locally modulate the activity of proteins involved in microtubule growth regulation and severing. Many of the proteins and mechanisms involved have roles in other microtubule assemblies as well, bestowing broader relevance to insights gained from plants.     

The ciliary membrane‐associated proteome reveals actin‐binding proteins as key components of cilia

Primary cilia are sensory, antennae‐like organelles present on the surface of many cell types. They have been involved in a variety of diseases collectively termed ciliopathies. As cilia are essential regulators of cell signaling, the composition of the ciliary membrane needs to be strictly regulated. To understand regulatory processes at the ciliary membrane, we report the targeting of a genetically engineered enzyme specifically to the ciliary membrane to allow biotinylation and identification of the membrane‐associated proteome. Bioinformatic analysis of the comprehensive dataset reveals high‐stoichiometric presence of actin‐binding proteins inside the cilium. Immunofluorescence stainings and complementary interaction proteomic analyses confirm these findings. Depolymerization of branched F‐actin causes further enrichment of the actin‐binding and actin‐related proteins in cilia, including Myosin 5a (Myo5a). Interestingly, Myo5a knockout decreases ciliation while enhanced levels of Myo5a are observed in cilia upon induction of ciliary disassembly. In summary, we present a novel approach to investigate dynamics of the ciliary membrane proteome in mammalian cells and identify actin‐binding proteins as mechanosensitive components of cilia that might have important functions in cilia membrane dynamics.     

Plant microtubule-associated proteins: the HEAT is off in temperature-sensitive mor1.

Microtubules perform essential functions in plant cells and govern, with other cytoskeletal elements, cell division, formation of cell walls and morphogenesis. For microtubules to perform their roles in the cell their organization and dynamics must be regulated and microtubule-associated proteins bear the main responsibility for these activities. We are just beginning to identify these plant microtubule-regulating proteins. Biochemical, molecular and genetic procedures have identified plant homologues of known microtubule-associated proteins, such as kinesins, katanin and XMAP215, and novel classes of plant microtubule-associated proteins, such as MAP65 and MAP190. Showing how these proteins coordinate the microtubule cytoskeleton in vivo is now the challenge. The recent identification and characterization of the Arabidopsis thaliana microtubule organization mutant, mor1, begins to address this challenge and here we highlight the significance of this work.     

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

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

Replication fork passage drives asymmetric dynamics of a critical nucleoid‐associated protein in Caulobacter

In bacteria, chromosome dynamics and gene expression are modulated by nucleoid‐associated proteins (NAPs), but little is known about how NAP activity is coupled to cell cycle progression. Using genomic techniques, quantitative cell imaging, and mathematical modeling, our study in Caulobacter crescentus identifies a novel NAP (GapR) whose activity over the cell cycle is shaped by DNA replication. GapR activity is critical for cellular function, as loss of GapR causes severe, pleiotropic defects in growth, cell division, DNA replication, and chromosome segregation. GapR also affects global gene expression with a chromosomal bias from origin to terminus, which is associated with a similar general bias in GapR binding activity along the chromosome. Strikingly, this asymmetric localization cannot be explained by the distribution of GapR binding sites on the chromosome. Instead, we present a mechanistic model in which the spatiotemporal dynamics of GapR are primarily driven by the progression of the replication forks. This model represents a simple mechanism of cell cycle regulation, in which DNA‐binding activity is intimately linked to the action of DNA replication.