Cyclic Nucleotide Gated Channels 7 and 8 Are Essential for Male Reproductive Fertility

The Arabidopsis thaliana genome contains 20 CNGCs, which are proposed to encode cyclic nucleotide gated, non-selective, Ca²⁺-permeable ion channels. CNGC7 and CNGC8 are the two most similar with 74% protein sequence identity, and both genes are preferentially expressed in pollen. Two independent loss-of-function T-DNA insertions were identified for both genes and used to generate plant lines in which only one of the two alleles was segregating (e.g., cngc7-1+/−/cngc8-2−/− and cngc7-3−/−/cngc8-1+/−). While normal pollen transmission was observed for single gene mutations, pollen harboring mutations in both cngc7 and 8 were found to be male sterile (transmission efficiency reduced by more than 3000-fold). Pollen grains harboring T-DNA disruptions of both cngc7 and 8 displayed a high frequency of bursting when germinated in vitro. The male sterile defect could be rescued through pollen expression of a CNGC7 or 8 transgene including a CNGC7 with an N-terminal GFP-tag. However, rescue efficiencies were reduced ∼10-fold when the CNGC7 or 8 included an F to W substitution (F589W and F624W, respectively) at the junction between the putative cyclic nucleotide binding-site and the calmodulin binding-site, identifying this junction as important for proper functioning of a plant CNGC. Using confocal microscopy, GFP-CNGC7 was found to preferentially localize to the plasma membrane at the flanks of the growing tip. Together these results indicate that CNGC7 and 8 are at least partially redundant and provide an essential function at the initiation of pollen tube tip growth.     

The Arabidopsis NUCLEUS- AND PHRAGMOPLAST-LOCALIZED KINASE1-Related Protein Kinases Are Required for Elicitor-Induced Oxidative Burst and Immunity

Plant immunity is activated through complex and cross-talking transduction pathways that include a mitogen-activated protein kinase phosphorylation cascade. Here, we have investigated the role in immunity of the Arabidopsis (Arabidopsis thaliana) gene subfamily that encodes the mitogen-activated protein triple kinases indicated as ARABIDOPSIS NUCLEUS- AND PHRAGMOPLAST-LOCALIZED KINASE1-RELATED PROTEIN KINASE1 (ANP1), ANP2, and ANP3. For this study, we used representative danger signals (elicitors) belonging to the classes of the damage- and pathogen-associated molecular patterns, i.e. oligogalacturonides, linear fragments derived from the plant cell wall homogalacturonan, and the peptide elf18 derived from the bacterial elongation factor thermo-unstable. Analyses of single and double as well as conditional triple mutants show that ANPs are required for elicitor-triggered defense responses and protection against the necrotrophic fungus Botrytis cinerea. Notably, ANPs are also required for both the elicitor-induced oxidative burst and the transduction of the hydrogen peroxide signal but not for the inhibition of auxin-induced gene expression, indicating that this response can be uncoupled from the activation of defense responses. Our findings point to ANPs as key transduction elements that coordinate damage- and pathogen-associated molecular pattern-triggered immunity and orchestrate reactive oxygen species accumulation and signaling.Plants are continually exposed to microbial pathogens and, like animals, activate the innate immune system to respond properly and in a timely manner (Boller and He, 2009). Plants also rely on the structure of the cell wall that acts as a physical barrier against the microbial invasion (De Lorenzo et al., 2001). In their attempts to penetrate plant tissues, pathogens need to efficiently degrade the cell wall (Lionetti et al., 2010). Once the plant cell wall is breached, pathogens encounter the host plasma membrane, where pattern recognition receptors (PRRs) sense the presence of nonself molecules (pathogen-associated molecular patterns [PAMPs]) and activate the so-called PAMP-triggered immunity (PTI; Dodds and Rathjen, 2010). Endogenous molecules, released during infection or mechanical wounding and usually referred to as damage-associated molecular patterns (DAMPs), are also recognized by PRRs as danger signals and contribute to the activation of the plant immune response (Schwessinger and Ronald, 2012). Representative PAMPs are the peptides elf18 and flg22, derived from the bacterial elongation factor thermo-unstable (EF-Tu) and flagellin, respectively (Gómez-Gómez and Boller, 2000; Zipfel et al., 2006). Among the best characterized DAMPs are oligogalacturonides (OGs), linear molecules of 10 to about 16 α-1,4-d-galactopyranosyluronic acid residues released upon fragmentation of homogalacturonan, which is an important component of the plant cell wall (Ferrari et al., 2013). In Arabidopsis (Arabidopsis thaliana), elf18 and flg22 are recognized by the transmembrane leucine-rich repeat receptor kinases EF-TU RECEPTOR (EFR) and FLAGELLIN-SENSING2, respectively (Gómez-Gómez and Boller, 2000; Zipfel et al., 2006). OGs, instead, are recognized by the WALL-ASSOCIATED KINASE1 (WAK1), a receptor kinase containing epidermal growth factor-like repeats (Brutus et al., 2010).Activation of PRRs leads to a prompt induction of ion fluxes, an oxidative burst, and defense gene expression and to late responses such as callose deposition, seedling growth inhibition, and protection against further pathogen attack. An overlap but also some distinctive features exist between responses induced by PAMPs and DAMPs. For example, flg22 and OGs generate an extracellular oxidative burst mediated by RESPIRATORY BURST OXIDASE HOMOLOG D (RbohD) and induce protection against the necrotrophic fungus Botrytis cinerea independently of the ethylene, jasmonic acid, and salicylic acid pathways and of the RbohD-mediated production of reactive oxygen species (ROS; Zhang et al., 2007; Galletti et al., 2008). The inhibition of auxin responses is another feature shared by PAMPs and DAMPs (Savatin et al., 2011); in the case of OGs, the inhibition of auxin responses has been described as a true antagonism (Branca et al., 1988; Bellincampi et al., 1993; Savatin et al., 2011). On the other hand, microarray analyses indicate that late responses to the two classes of elicitors are considerably different (Denoux et al., 2008).In plants, as in animals, immunity is activated through complex and cross-talking transduction pathways that include a mitogen-activated protein (MAP) kinase (MAPK) phosphorylation cascade (Rodriguez et al., 2010). A MAPK cascade consists of a core module of three kinases that perform sequential phosphorylation reactions: a MAP kinase kinase kinase (MAP3K) activates, by phosphorylation, a MAP kinase kinase (MAP2K), which activates a MAPK. Sixty MAP3Ks, 10 MAP2Ks, and 20 MAPKs are encoded by the Arabidopsis genome (Ichimura et al., 2002), leading to a complexity that hampers the characterization of this transduction system. Three immune-related MAPK modules have been identified. A module comprising the MAP3K MEKK1, the MAP2Ks MKK1 and MKK2, and the MAPK MPK4 (MEKK1/MKK1-MKK2/MPK4) negatively controls defense responses (Kong et al., 2012; Rasmussen et al., 2012) by negatively regulating the expression of the MAP3K MEKK2, which triggers a salicylate (SA)-dependent autoimmunity response when the cascade is compromised (Berriri et al., 2012; Su et al., 2013). Two other modules, MEKK1-MAPKKKα/MKK4-MKK5/MPK3-MPK6 (Ren et al., 2008) and MKK9/MPK3-MPK6 (Xu et al., 2008), mediate the activation of defense responses, including the synthesis of ethylene and camalexin, i.e. a phytoalexin with antimicrobial activity. The only MAPK elements shown so far to participate in the response to DAMPs are the Arabidopsis MPK3 and MPK6. Both are phosphorylated within minutes upon elicitation with OGs and flg22, but only MPK6 is required for full elicitor-induced up-regulation of defense genes and protection against B. cinerea (Galletti et al., 2011).The subfamily of MAP3Ks indicated as ARABIDOPSIS NUCLEUS- AND PHRAGMOPLAST-LOCALIZED KINASE1 (NPK1)-RELATED PROTEIN KINASEs (ANPs) includes three members, ANP1, ANP2, and ANP3, that were initially identified for their homology with the tobacco (Nicotiana tabacum) NPK1 (Jouannic et al., 1999; Sasabe and Machida, 2012). NPK1 regulates cytokinesis (Nakashima et al., 1998) as well as the effector-triggered immunity and development in Nicotiana benthamiana (Jin et al., 2002). ANPs have also been reported to be involved in the inhibition of auxin-induced gene expression. Constitutively active forms of ANPs, obtained by deletion of the C-terminal regulatory domain (ΔANPs) and expressed in protoplasts, negatively regulate the activity of the auxin-inducible soybean (Glycine max) GRETCHEN HAGEN3 promoter and activate the hydrogen peroxide signaling pathway (Kovtun et al., 2000). Therefore, ANPs have been proposed as a molecular link between oxidative stress and auxin signal transduction. However, we have previously shown that double knockout (KO) anp mutants exhibit a normal auxin/OG antagonism (Savatin et al., 2011), thus providing no support to this conclusion. Our work left unanswered whether this was due to the presence of a functional third ANP member or to a lack of involvement of ANPs in elicitor/auxin antagonism and, in general, in the response to OGs, because a main role of ANP1 in response to flg22 had been previously ruled out (Asai et al., 2002).The anp2 anp3 double mutant displays developmental defects related to cytokinesis as well as up-regulation of stress-related genes, while an anp triple KO mutant was never obtained, probably because ANPs are essential for plant development (Krysan et al., 2002). The phenotype of the anp2 anp3 mutant is similar to that of mpk4 mutant, suggesting that ANPs and MPK4 may be part of the same transduction pathway and act as negative regulators of defense responses (Beck et al., 2011).We show here that ANP genes are not involved in elicitor-auxin antagonism but are required for DAMP and PAMP signal transduction. Single and double mutants as well as conditional triple mutants, which were generated in this work, are defective in defense responses to both OGs and elf18. Notably, ANPs are required for both elicitor-induced generation of ROS and response to ROS. Our study points to ANPs as key regulators of plant immunity.     

PTK7/Otk interacts with Wnts and inhibits canonical Wnt signalling

Wnt signalling is an evolutionarily conserved pathway that directs cell-fate determination and morphogenesis during metazoan development. Wnt ligands are secreted glycoproteins that act at a distance causing a wide range of cellular responses from stem cell maintenance to cell death and cell proliferation. How Wnt ligands cause such disparate responses is not known, but one possibility is that different outcomes are due to different receptors. Here, we examine PTK7/Otk, a transmembrane receptor that controls a variety of developmental and physiological processes including the regulation of cell polarity, cell migration and invasion. PTK7/Otk co-precipitates canonical Wnt3a and Wnt8, indicating a role in Wnt signalling, but PTK7 inhibits rather than activates canonical Wnt activity in Xenopus, Drosophila and luciferase reporter assays. Loss of PTK7 function activates canonical Wnt signalling and epistasis experiments place PTK7 at the level of the Frizzled receptor. In Drosophila, Otk interacts with Wnt4 and opposes canonical Wnt signalling in embryonic patterning. We propose a model where PTK7/Otk functions in non-canonical Wnt signalling by turning off the canonical signalling branch.     

Uclacyanin Proteins Are Required for Lignified Nanodomain Formation within Casparian Strips

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Host ESCRT Proteins Are Required for Bromovirus RNA Replication Compartment Assembly and Function

Positive-strand RNA viruses genome replication invariably is associated with vesicles or other rearranged cellular membranes. Brome mosaic virus (BMV) RNA replication occurs on perinuclear endoplasmic reticulum (ER) membranes in ~70 nm vesicular invaginations (spherules). BMV RNA replication vesicles show multiple parallels with membrane-enveloped, budding retrovirus virions, whose envelopment and release depend on the host ESCRT (endosomal sorting complexes required for transport) membrane-remodeling machinery. We now find that deleting components of the ESCRT pathway results in at least two distinct BMV phenotypes. One group of genes regulate RNA replication and the frequency of viral replication complex formation, but had no effect on spherule size, while a second group of genes regulate RNA replication in a way or ways independent of spherule formation. In particular, deleting SNF7 inhibits BMV RNA replication > 25-fold and abolishes detectable BMV spherule formation, even though the BMV RNA replication proteins accumulate and localize normally on perinuclear ER membranes. Moreover, BMV ESCRT recruitment and spherule assembly depend on different sets of protein-protein interactions from those used by multivesicular body vesicles, HIV-1 virion budding, or tomato bushy stunt virus (TBSV) spherule formation. These and other data demonstrate that BMV requires cellular ESCRT components for proper formation and function of its vesicular RNA replication compartments. The results highlight growing but diverse interactions of ESCRT factors with many viruses and viral processes, and potential value of the ESCRT pathway as a target for broad-spectrum antiviral resistance.     

LRGUK-1 Is Required for Basal Body and Manchette Function during Spermatogenesis and Male Fertility

Male infertility affects at least 5% of reproductive age males. The most common pathology is a complex presentation of decreased sperm output and abnormal sperm shape and motility referred to as oligoasthenoteratospermia (OAT). For the majority of OAT men a precise diagnosis cannot be provided. Here we demonstrate that leucine-rich repeats and guanylate kinase-domain containing isoform 1 (LRGUK-1) is required for multiple aspects of sperm assembly, including acrosome attachment, sperm head shaping and the initiation of the axoneme growth to form the core of the sperm tail. Specifically, LRGUK-1 is required for basal body attachment to the plasma membrane, the appropriate formation of the sub-distal appendages, the extension of axoneme microtubules and for microtubule movement and organisation within the manchette. Manchette dysfunction leads to abnormal sperm head shaping. Several of these functions may be achieved in association with the LRGUK-1 binding partner HOOK2. Collectively, these data establish LRGUK-1 as a major determinant of microtubule structure within the male germ line.     

Outer Membrane Proteins Ail and OmpF of Yersinia pestis Are Involved in the Adsorption of T7-Related Bacteriophage Yep-phi

Yep-phi is a T7-related bacteriophage specific to Yersinia pestis, and it is routinely used in the identification of Y. pestis in China. Yep-phi infects Y. pestis grown at both 20°C and 37°C. It is inactive in other Yersinia species irrespective of the growth temperature. Based on phage adsorption, phage plaque formation, affinity chromatography, and Western blot assays, the outer membrane proteins of Y. pestis Ail and OmpF were identified to be involved, in addition to the rough lipopolysaccharide, in the adsorption of Yep-phi. The phage tail fiber protein specifically interacts with Ail and OmpF proteins, and residues 518N, 519N, and 523S of the phage tail fiber protein are essential for the interaction with OmpF, whereas residues 518N, 519N, 522C, and 523S are essential for the interaction with Ail. This is the first report to demonstrate that membrane-bound proteins are involved in the adsorption of a T7-related bacteriophage. The observations highlight the importance of the tail fiber protein in the evolution and function of various complex phage systems and provide insights into phage-bacterium interactions.     

Whirlin and PDZ Domain-containing 7 (PDZD7) Proteins Are Both Required to Form the Quaternary Protein Complex Associated with Usher Syndrome Type 2

Usher syndrome (USH) is the leading genetic cause of combined hearing and vision loss. Among the three USH clinical types, type 2 (USH2) occurs most commonly. USH2A, GPR98, and WHRN are three known causative genes of USH2, whereas PDZD7 is a modifier gene found in USH2 patients. The proteins encoded by these four USH genes have been proposed to form a multiprotein complex, the USH2 complex, due to interactions found among some of these proteins in vitro, their colocalization in vivo, and mutual dependence of some of these proteins for their normal in vivo localizations. However, evidence showing the formation of the USH2 complex is missing, and details on how this complex is formed remain elusive. Here, we systematically investigated interactions among the intracellular regions of the four USH proteins using colocalization, yeast two-hybrid, and pull-down assays. We show that multiple domains of the four USH proteins interact among one another. Importantly, both WHRN and PDZD7 are required for the complex formation with USH2A and GPR98. In this USH2 quaternary complex, WHRN prefers to bind to USH2A, whereas PDZD7 prefers to bind to GPR98. Interaction between WHRN and PDZD7 is the bridge between USH2A and GPR98. Additionally, the USH2 quaternary complex has a variable stoichiometry. These findings suggest that a non-obligate, short term, and dynamic USH2 quaternary protein complex may exist in vivo. Our work provides valuable insight into the physiological role of the USH2 complex in vivo and informs possible reconstruction of the USH2 complex for future therapy.     

The HOPS Proteins hVps41 and hVps39 Are Required for Homotypic and Heterotypic Late Endosome Fusion

The homotypic fusion and protein sorting (HOPS) complex is a multisubunit tethering complex that in yeast regulates membrane fusion events with the vacuole, the yeast lysosome. Mammalian homologs of all HOPS components have been found, but little is known about their function. Here, we studied the role of hVps41 and hVps39, two components of the putative human HOPS complex, in the endo‐lysosomal pathway of human cells. By expressing hemagglutinin (HA)‐tagged constructs, we show by immunoelectron microscopy (immunoEM) that both hVps41 and hVps39 associate with the limiting membrane of late endosomes as well as lysosomes. Small interference RNA (siRNA)‐mediated knockdown of hVps41 or hVps39 resulted in an accumulation of late endosomes, a depletion in the number of lysosomes and a block in the degradation of endocytosed cargo. Lysosomal pH and cathepsin B activity remained unaltered in these conditions. By immunoEM we found that hVps41 or hVps39 knockdown impairs homotypic fusion between late endosomes as well as heterotypic fusion between late endosomes and lysosomes. Thus, our data show that both hVps41 and hVps39 are required for late endosomal–lysosomal fusion events and the delivery of endocytic cargo to lysosomes in human cells.     

The Genes Raw and Ribbon Are Required for Proper Shape of Tubular Epithelial Tissues in Drosophila

The products of two genes, raw and ribbon (rib), are required for the proper morphogenesis of a variety of tissues. Malpighian tubules mutant for raw or rib are wider and shorter than normal tubules, which are only two cells in circumference when they are fully formed. The mutations alter the shape of the tubules beginning early in their formation and block cell rearrangement late in development, which normally lengthens and narrows the tubes. Mutations of both genes affect a number of other tissues as well. Both genes are required for dorsal closure and retraction of the CNS during embryonic development. In addition, rib mutations block head involution, and broaden and shorten other tubular epithelia (salivary glands, tracheae, and hindgut) in much same manner as they alter the shape of the Malpighian tubules. In tissues in which the shape of cells can be observed readily, rib mutations alter cell shape, which probably causes the change in shape of the organs that are affected. In double mutants raw enhances the phenotypes of all the tissues that are affected by rib but unaffected by raw alone, indicating that raw is also active in these tissues.     

MreC and MreD Proteins Are Not Required for Growth of Staphylococcus aureus

The transmembrane proteins MreC and MreD are present in a wide variety of bacteria and are thought to be involved in cell shape determination. Together with the actin homologue MreB and other morphological elements, they play an essential role in the synthesis of the lateral cell wall in rod-shaped bacteria. In ovococcus, which lack MreB homologues, mreCD are also essential and have been implicated in peripheral cell wall synthesis. In this work we addressed the possible roles of MreC and MreD in the spherical pathogen Staphylococcus aureus. We show that MreC and MreD are not essential for cell viability and do not seem to affect cell morphology, cell volume or cell cycle control. MreC and MreD localize preferentially to the division septa, but do not appear to influence peptidoglycan composition, nor the susceptibility to different antibiotics and to oxidative and osmotic stress agents. Our results suggest that the function of MreCD in S. aureus is not critical for cell division and cell shape determination.     

Drosophila Centrosomin Protein is Required for Male Meiosis and Assembly of the Flagellar Axoneme

Centrosomes and microtubules play crucial roles during cell division and differentiation. Spermatogenesis is a useful system for studying centrosomal function since it involves both mitosis and meiosis, and also transformation of the centriole into the sperm basal body. Centrosomin is a protein localized to the mitotic centrosomes in Drosophila melanogaster. We have found a novel isoform of centrosomin expressed during spermatogenesis. Additionally, an anticentrosomin antibody labels both the mitotic and meiotic centrosomes as well as the basal body. Mutational analysis shows that centrosomin is required for spindle organization during meiosis and for organization of the sperm axoneme. These results suggest that centrosomin is a necessary component of the meiotic centrosomes and the spermatid basal body.     

E-Cadherin Is Required for Centrosome and Spindle Orientation in Drosophila Male Germline Stem Cells

Many adult stem cells reside in a special microenvironment known as the niche, where they receive essential signals that specify stem cell identity. Cell-cell adhesion mediated by cadherin and integrin plays a crucial role in maintaining stem cells within the niche. In Drosophila melanogaster, male germline stem cells (GSCs) are attached to niche component cells (i.e., the hub) via adherens junctions. The GSC centrosomes and spindle are oriented toward the hub-GSC junction, where E-cadherin-based adherens junctions are highly concentrated. For this reason, adherens junctions are thought to provide a polarity cue for GSCs to enable proper orientation of centrosomes and spindles, a critical step toward asymmetric stem cell division. However, understanding the role of E-cadherin in GSC polarity has been challenging, since GSCs carrying E-cadherin mutations are not maintained in the niche. Here, we tested whether E-cadherin is required for GSC polarity by expressing a dominant-negative form of E-cadherin. We found that E-cadherin is indeed required for polarizing GSCs toward the hub cells, an effect that may be mediated by Apc2. We also demonstrated that E-cadherin is required for the GSC centrosome orientation checkpoint, which prevents mitosis when centrosomes are not correctly oriented. We propose that E-cadherin orchestrates multiple aspects of stem cell behavior, including polarization of stem cells toward the stem cell-niche interface and adhesion of stem cells to the niche supporting cells.     

The Nek6 and Nek7 Protein Kinases Are Required for Robust Mitotic Spindle Formation and Cytokinesis

Nek6 and Nek7 are members of the NIMA-related serine/threonine kinase family. Previous work showed that they contribute to mitotic progression downstream of another NIMA-related kinase, Nek9, although the roles of these different kinases remain to be defined. Here, we carried out a comprehensive analysis of the regulation and function of Nek6 and Nek7 in human cells. By generating specific antibodies, we show that both Nek6 and Nek7 are activated in mitosis and that interfering with their activity by either depletion or expression of reduced-activity mutants leads to mitotic arrest and apoptosis. Interestingly, while completely inactive mutants and small interfering RNA-mediated depletion delay cells at metaphase with fragile mitotic spindles, hypomorphic mutants or RNA interference treatment combined with a spindle assembly checkpoint inhibitor delays cells at cytokinesis. Importantly, depletion of either Nek6 or Nek7 leads to defective mitotic progression, indicating that although highly similar, they are not redundant. Indeed, while both kinases localize to spindle poles, only Nek6 obviously localizes to spindle microtubules in metaphase and anaphase and to the midbody during cytokinesis. Together, these data lead us to propose that Nek6 and Nek7 play independent roles not only in robust mitotic spindle formation but also potentially in cytokinesis.When cells divide, they must accurately segregate the duplicated genetic material between two daughter cells such that each receives a single complete set of chromosomes. This complex biomechanical feat is achieved through the action of a bipolar microtubule-based scaffold called the mitotic spindle (36). Microtubules are primarily nucleated by centrosomes that sit at the spindle poles (37). However, microtubule nucleation also occurs in the vicinity of the chromosomes and within the spindle itself (12, 13). These activities combine to ensure the efficient capture of sister chromatids as well as the maintenance of a robust structure capable of resisting the considerable forces required for chromosome separation.Spindle assembly is regulated in large part by reversible phosphorylation, and a number of protein kinases are activated during mitosis, localize to specific regions of the spindle, and phosphorylate spindle-associated proteins. These include the master mitotic regulator Cdk1/cyclin B, the polo-like kinase Plk1, and the Aurora family kinases Aurora A and B (25). More recently, members of the NIMA-related kinase family have also been implicated in mitotic spindle regulation (27, 29). NIMA was first identified in Aspergillus nidulans as a kinase required for mitotic entry, possibly through triggering the relocation of Cdk1/cyclin B to the nucleus (6, 38). NIMA can also phosphorylate S10 of histone H3 to promote chromatin condensation (7). The fission yeast NIMA-related kinase Fin1 contributes to multiple steps in mitotic progression, including the timing of mitotic entry, spindle formation, and mitotic exit (14, 15). However, the detailed mechanisms by which these fungal kinases contribute to mitotic regulation remain far from understood.In mammals, there are 11 NIMA-related kinases, named Nek1 to Nek11, and of these, 4 have been directly implicated in mitotic regulation, as follows: Nek2, Nek6, Nek7, and Nek9 (also known as Nercc1) (26, 27, 29). Nek2 is the most closely related mammalian kinase to NIMA and Fin1 by sequence and has been studied in the most detail. It localizes to the centrosome, where it phosphorylates and thereby regulates the association of a number of large coiled-coil proteins implicated in centrosome cohesion and microtubule anchoring (1, 10, 11, 21, 22, 30). These activities facilitate the early stages of spindle assembly at the G₂/M transition. Interestingly, Aspergillus NIMA and fission yeast Fin1 also localize to the fungal equivalent of the centrosome, namely the spindle pole body (15, 20, 38). Here, they may participate in positive feedback loops that promote the activation of Cdk1/cyclin B and mitotic entry.Nek6, Nek7, and Nek9 act together in a mitotic kinase cascade, with Nek9 being upstream of Nek6 and Nek7. Nek9 was identified as an interacting partner of Nek6 and subsequently shown to phosphorylate Nek6 at S206 within its activation loop (2, 33). Both Nek9 and Nek6 have been reported to be activated in mitosis (2, 33, 39), although other studies dispute this (18, 23). NIMA-related kinases are characterized by having a conserved N-terminal catalytic domain, followed by a nonconserved C-terminal regulatory domain that varies in size and structure. Nek6 and Nek7 are significant exceptions to this, in that they are the smallest of the kinases and consist only of a catalytic domain with a very short N-terminal extension. They share significant similarity with each other, being 87% identical within their catalytic domains. Hence, although they exhibit distinct tissue expression patterns (8), it has generally been assumed that they are likely to have very similar properties and functions, with both being downstream substrates of Nek9.Functional studies of Nek9 reveal that it has major roles to play in the organization of the mitotic spindle. Expression of inactive and truncated Nek9 mutants led to the missegregation of chromosomes, while injection of anti-Nek9 antibodies into prophase cells caused aberrant mitotic spindle formation (33). Similarly, depletion of Nek9 from Xenopus egg extracts led to a reduction in the formation of bipolar spindles in vitro (32; J. Blot and A. M. Fry, unpublished results). The basis for these defects remains unclear, but a number of binding partners have been identified that suggest possible functions in microtubule nucleation and anchoring, including components of the γ-tubulin ring complex (γ-TuRC), the Ran GTPase, and BicD2 (18, 32, 33).While Nek9 is proposed to act upstream of Nek6 and Nek7, the proportion of its activities being channeled through these kinases is not known. Limited studies have been performed by looking at the consequences of expressing kinase-inactive Nek6 or Nek7 constructs or depleting the proteins by RNA interference (RNAi). Interference with Nek6 has been reported by one group to lead to metaphase arrest and apoptosis (39), although this is disputed by another study (23). Interference with Nek7 apparently leads to an increase in the mitotic index and apoptosis (19, 40). A decrease in centrosome-associated γ-tubulin and microtubule nucleation was also detected upon RNAi of Nek7, which is interesting in light of the interaction between Nek9 and γ-tubulin. Furthermore, defects in cytokinesis were found upon Nek7 depletion if cells were allowed to progress past the spindle checkpoint by codepletion of Mad2 (19). Importantly, both Nek9 and Nek7 localize to centrosomes, further supporting the model that this is a major site of action for this family of kinases in spindle formation (19, 32, 40).In this study, we set out to clarify the mitotic roles of Nek6 and Nek7 by examining the consequences of expression of mutants with different levels of kinase activity as well as depletion of the proteins by RNAi. Our results demonstrate that Nek6 and Nek7 are both activated in mitosis and that interference with either kinase leads to apoptosis following mitotic arrest. Interestingly, expression of inactive mutants or small interfering RNA (siRNA)-mediated depletion leads to a metaphase delay with fragile mitotic spindles, whereas expression of hypomorphic mutants or depletion in the presence of a spindle assembly checkpoint (SAC) inhibitor leads to an accumulation of cells in cytokinesis. Based on additional localization data, we propose that these kinases regulate microtubule organization not only at spindle poles but also within the mitotic spindle itself and possibly at the central spindle during late mitosis. This study therefore provides important novel insights into how Nek6 and Nek7 contribute to distinct molecular events in mitotic progression.