Absence of integrin alpha 7 causes a novel form of muscular dystrophy.

Integrin alpha 7 beta 1 is a specific cellular receptor for the basement membrane protein laminin-1 (refs 1,2), as well as for the laminin isoforms -2 and -4 (ref. 3). The alpha 7 subunit is expressed mainly in skeletal and cardiac muscle and has been suggested to be involved in differentiation and migration processes during myogenesis. Three cytoplasmic and two extracellular splice variants that have been described are developmentally regulated and expressed in different sites in the muscle. In adult muscle, the alpha 7A and alpha 7B subunits are concentrated in myotendinous junctions but can also be detected in neuromuscular junctions and along the sarcolemmal membrane. To study the potential involvement of alpha 7 integrin, during myogenesis and its role in muscle integrity and function, we generated a null allele of the alpha 7 gene (Itga7) in the germline of mice by homologous recombination in embryonic stem (ES) cells. Surprisingly, mice homozygous for the mutation are viable and fertile, indicating that the alpha 7 beta 1 integrin is not essential for myogenesis. However, histological analysis of skeletal muscle revealed typical symptoms of a progressive muscular dystrophy starting soon after birth, but with a distinct variability in different muscle types. The observed histopathological changes strongly indicate an impairment of function of the myotendinous junctions. These findings demonstrate that alpha 7 beta 1 integrin represents an indispensable linkage between the muscle fibre and the extracellular matrix that is independent of the dystrophin-dystroglycan complex-mediated interaction of the cytoskeleton with the muscle basement membrane.

Increased sensitivity to anticancer drugs and decreased inflammatory response in mice lacking the multidrug resistance-associated protein.

The multidrug resistance-associated protein (MRP) mediates the cellular excretion of many drugs, glutathione S-conjugates (GS-X) of lipophilic xenobiotics and endogenous cysteinyl leukotrienes. Increased MRP levels in tumor cells can cause multidrug resistance (MDR) by decreasing the intracellular drug concentration. The physiological role or roles of MRP remain ill-defined, however. We have generated MRP-deficient mice by using embryonic stem cell technology. Mice homozygous for the mrp mutant allele, mrp-/-, are viable and fertile, but their response to an inflammatory stimulus is impaired. We attribute this defect to a decreased secretion of leukotriene C4 (LTC4) from leukotriene-synthesizing cells. Moreover, the mrp-/- mice are hypersensitive to the anticancer drug etoposide. The phenotype of mrp-/- mice is consistent with a role for MRP as the main LTC4-exporter in leukotriene-synthesizing cells, and as an important drug exporter in drug-sensitive cells. Our results suggest that this ubiquitous GS-X pump is dispensable in mice, making treatment of MDR with MRP-specific reversal agents potentially feasible.

Regulatory network analysis of Paneth cell and goblet cell enriched gut organoids using transcriptomics approaches.

The epithelial lining of the small intestine consists of multiple cell types, including Paneth cells and goblet cells, that work in cohort to maintain gut health. 3D in vitro cultures of human primary epithelial cells, called organoids, have become a key model to study the functions of Paneth cells and goblet cells in normal and diseased conditions. Advances in these models include the ability to skew differentiation to particular lineages, providing a useful tool to study cell type specific function/dysfunction in the context of the epithelium. Here, we use comprehensive profiling of mRNA, microRNA and long non-coding RNA expression to confirm that Paneth cell and goblet cell enrichment of murine small intestinal organoids (enteroids) establishes a physiologically accurate model. We employ network analysis to infer the regulatory landscape altered by skewing differentiation, and using knowledge of cell type specific markers, we predict key regulators of cell type specific functions: Cebpa, Jun, Nr1d1 and Rxra specific to Paneth cells, Gfi1b and Myc specific for goblet cells and Ets1, Nr3c1 and Vdr shared between them. Links identified between these regulators and cellular phenotypes of inflammatory bowel disease (IBD) suggest that global regulatory rewiring during or after differentiation of Paneth cells and goblet cells could contribute to IBD aetiology. Future application of cell type enriched enteroids combined with the presented computational workflow can be used to disentangle multifactorial mechanisms of these cell types and propose regulators whose pharmacological targeting could be advantageous in treating IBD patients with Crohn's disease or ulcerative colitis.

The cytokinesis gene KEULE encodes a Sec1 protein that binds the syntaxin KNOLLE.

KEULE is required for cytokinesis in Arabidopsis thaliana. We have positionally cloned the KEULE gene and shown that it encodes a Sec1 protein. KEULE is expressed throughout the plant, yet appears enriched in dividing tissues. Cytokinesis-defective mutant sectors were observed in all somatic tissues upon transformation of wild-type plants with a KEULE-green fluorescent protein gene fusion, suggesting that KEULE is required not only during embryogenesis, but at all stages of the plant's life cycle. KEULE is characteristic of a Sec1 protein in that it appears to exist in two forms: soluble or peripherally associated with membranes. More importantly, KEULE binds the cytokinesis-specific syntaxin KNOLLE. Sec1 proteins are key regulators of vesicle trafficking, capable of integrating a large number of intra- and/or intercellular signals. As a cytokinesis-related Sec1 protein, KEULE appears to represent a novel link between cell cycle progression and the membrane fusion apparatus.

The Arabidopsis KNOLLE protein is a cytokinesis-specific syntaxin.

In higher plant cytokinesis, plasma membrane and cell wall originate by vesicle fusion in the plane of cell division. The Arabidopsis KNOLLE gene, which is required for cytokinesis, encodes a protein related to vesicle-docking syntaxins. We have raised specific rabbit antiserum against purified recombinant KNOLLE protein to show biochemically and by immunoelectron microscopy that KNOLLE protein is membrane associated. Using immunofluorescence microscopy, KNOLLE protein was found to be specifically expressed during mitosis and, unlike the plasma membrane H+-ATPase, to localize to the plane of division during cytokinesis. Arabidopsis dynamin-like protein ADL1 accumulates at the plane of cell plate formation in knolle mutant cells as in wild-type cells, suggesting that cytokinetic vesicle traffic is not affected. Furthermore, electron microscopic analysis indicates that vesicle fusion is impaired. KNOLLE protein was detected in mitotically dividing cells of various parts of the developing plant, including seedling root, inflorescence meristem, floral meristems and ovules, and the cellularizing endosperm, but not during cytokinesis after the male second meiotic division. Thus, KNOLLE is the first syntaxin-like protein that appears to be involved specifically in cytokinetic vesicle fusion.

Calcium-signaling networks in olfactory receptor neurons.

The olfactory neuroepithelium represents a unique interface between the brain and the external environment. Olfactory function comprises a distinct set of molecular tasks: sensory signal transduction, cytoprotection and adult neurogenesis. A multitude of biochemical studies has revealed the central role of Ca(2+) signaling in the function of olfactory receptor neurons (ORNs). We set out to establish Ca(2+)-dependent signaling networks in ORN cilia by proteomic analysis. We subjected a ciliary membrane preparation to Ca(2+)/calmodulin-affinity chromatography using mild detergent conditions in order to maintain functional protein complexes involved in olfactory Ca(2+) signaling. Thus, calmodulin serves as a valuable tool to gain access to novel Ca(2+)-regulated protein complexes. Tandem mass spectrometry (nanoscale liquid-chromatography-electrospray injection) identified 123 distinct proteins. Ninety-seven proteins (79%) could be assigned to specific olfactory functions, including 32 to sensory signal transduction and 40 to cytoprotection. We point out novel perspectives for research on the Ca(2+)-signaling networks in the olfactory system of the rat.

The Arabidopsis KNOLLE and KEULE genes interact to promote vesicle fusion during cytokinesis.

Partitioning of the cytoplasm during cytokinesis or cellularisation requires syntaxin-mediated membrane fusion [1-3]. Whereas in animals, membrane fusion promotes ingression of a cleavage furrow from the plasma membrane [4,5], somatic cells of higher plants form de novo a transient membrane compartment, the cell plate, which is initiated in the centre of the division plane and matures into a new cell wall and its flanking plasma membranes [6,7]. Cell plate formation results from the fusion of Golgi-derived vesicles delivered by a dynamic cytoskeletal array, the phragmoplast. Mutations in two Arabidopsis genes, KNOLLE (KN) and KEULE (KEU), cause abnormal seedlings with multinucleate cells and incomplete cell walls [1,8]. The KN gene encodes a cytokinesis-specific syntaxin which localises to the cell plate [9]. Here, we show that KN protein localisation is unaffected in keu mutant cells, which, like kn, display phragmoplast microtubules and accumulate ADL1 protein in the plane of cell division but vesicles fail to fuse with one another. Genetic interactions between KN and KEU were analysed in double mutant embryos. Whereas the haploid gametophytes gave rise to functional gametes, the embryos behaved like single cells displaying multiple, synchronously cycling nuclei, cell cycle-dependent microtubule arrays and ADL1 accumulation between pairs of daughter nuclei. This complete inhibition of cytokinesis from fertilisation indicates that KN and KEU, have partially redundant functions and interact specifically in vesicle fusion during cytokinesis of somatic cells.

Full blockade of intestinal P-glycoprotein and extensive inhibition of blood-brain barrier P-glycoprotein by oral treatment of mice with PSC833.

Mice lacking mdr1-type P-glycoproteins (mdr1a/1b [-/-] mice) display large changes in the pharmacokinetics of digoxin and other drugs. Using the kinetics of digoxin in mdr1a/1b (-/-) mice as a model representing a complete block of P-glycoprotein activity, we investigated the activity and specificity of the reversal agent SDZ PSC833 in inhibiting mdr1-type P-glycoproteins in vivo. Oral PSC833 was coadministered with intravenous [3H]digoxin to wild-type and mdr1a/1b (-/-) mice. The direct excretion of [3H]digoxin mediated by P-glycoprotein in the intestinal mucosa of wild-type mice was abolished by administration of PSC833. Hepatobiliary excretion of [3H]digoxin was markedly decreased in both wild-type and mdr1a/1b (-/-) mice by PSC833, the latter effect indicating that in vivo, PSC833 inhibits not only mdr1-type P-glycoproteins, but also other drug transporters. Upon coadministration of PSC833, brain levels of [3H]digoxin in wild-type mice showed a large increase, approaching (but not equaling) the levels found in brains of PSC833-treated mdr1a/1b (-/-) mice. Thus, orally administered PSC833 can inhibit blood-brain barrier P-glycoprotein extensively, and intestinal P-glycoprotein completely. These profound pharmacokinetic effects of PSC833 treatment imply potential risks, but also promising pharmacological applications of the use of effective reversal agents.

Pattern Formation in the Arabidopsis Embryo Revealed by Position-Specific Lipid Transfer Protein Gene Expression.

During Arabidopsis embryogenesis, the zygote divides asymmetrically in the future apical-basal axis; however, a radial axis is initiated only within the eight-celled embryo. Mutations in the GNOM, KNOLLE, and KEULE genes affect these processes: gnom zygotes tend to divide symmetrically; knolle embryos lack oriented cell divisions that initiate protoderm formation; and in keule embryos, an outer cell layer is present that consists of abnormally enlarged cells from early development. Pattern formation along the two axes is reflected by the position-specific expression of the Arabidopsis lipid transfer protein (AtLTP1) gene. In wild-type embryos, the AtLTP1 gene is expressed in the protoderm and initially in all protodermal cells; later, AtLTP1 expression is confined to the cotyledons and the upper end of the hypocotyl. Analysis of AtLTP1 expression in gnom, knolle, and keule embryos showed that gnom embryos also can have no or reversed apical-basal polarity, whereas radial polarity is unaffected. knolle embryos initially lack but eventually form a radial pattern, and keule embryos are affected in protoderm cell morphology rather than in the establishment of the radial pattern.

Cytokinesis in flowering plants: cellular process and developmental integration.

In phragmoplast-assisted cytokinesis of somatic cells, vesicle fusion generates a cell plate that matures into a new cell wall and its flanking plasma membranes. Insight into this dynamic process has been gained in the past few years and additional molecular components of the basic machinery of cytokinesis have been identified. Specialized modes of cytokinesis occur in meiosis and gametophyte development, and recent studies indicate that they are genetically distinct from somatic cytokinesis.

Structure and multiple conformations of the kunitz-type domain from human type VI collagen alpha3(VI) chain in solution.

BACKGROUND: The Kunitz-type inhibitor motif is found at the C terminus of the human collagen alpha3(VI) chain. This 76-residue module (domain C5) was prepared in recombinant form and showed high stability against proteases; however, it lacked any inhibitory activity against trypsin, thrombin, kallikrein and several other proteases. We have undertaken the determination of the three-dimensional (3D) structure of domain C5 in solution, by nuclear magnetic resonance (NMR), in order to establish the structural basis for the properties of this protein. RESULTS: The 7 N-terminal and 12 C-terminal residues of domain C5 are disordered in the solution structure. The 55-residue core, which shows high homology to bovine pancreatic trypsin inhibitor, retains the characteristic fold of all members of the Kunitz-type inhibitor family. 24 residues of this main structural body show more than one resonance, symptomatic of multiple conformations slowly exchanging on the NMR time scale. In addition, significant proton chemical exchange line broadening is observed for residues in the vicinity of the disulfide bridge between residues 20 and 44: this indicates interconversion, on the micro- to millisecond time scale, between multiple conformations. CONCLUSION: The NMR study demonstrates that domain C5 is a highly dynamic molecule at temperatures studied (between 10 and 30 degrees C). Indeed, some 44% of the main body structure of C5 showed multiple conformations. The existence of multiple conformations was not necessarily expected in view of the conformational constraints imposed by the 3D structure of proteins as rigid as C5; it should therefore be considered in the interpretation of its structural and dynamical properties. The accessibility of the inhibitory binding loop (Gly18 [P4] to Leu25 [P4']) should be relatively unaffected by this conformational exchange and thus would not explain the unusual specificity of C5. Most serine proteinase inhibitors that, like C5, have an arginine at the P1 position inhibit trypsin; the lack of trypsin inhibition of C5 must therefore arise from the amino-acid side-chain composition of the adjoining positions in the binding loop.

Availability of PSC833, a substrate and inhibitor of P-glycoproteins, in various concentrations of serum.

BACKGROUND: P-glycoproteins are membrane-associated transporters that can render cells resistant to a variety of chemotherapeutic drugs. Reversal agents are (preferably nontoxic) drugs that can inhibit these P-glycoproteins and thereby overcome multidrug resistance. PSC833, a cyclosporin A analog, is a reversal agent that has shown potential in in vitro experiments and in clinical trials. We tested PSC833 to determine whether it is a transported substrate of human and murine P-glycoproteins associated with multidrug resistance (encoded by the human MDR1 gene and its murine homolog, mdr1a) and whether it can completely inhibit these P-glycoproteins under simulated in vivo conditions. METHODS: Monolayers of polarized LLC-PK1 pig kidney cells transfected with complementary DNA containing either MDR1 or mdr1a sequences were used to measure the directional transport of P-glycoprotein substrates under various serum conditions. RESULTS: In contrast to two previous studies, we found that PSC833 is transported by both the MDR1 and the mdr1a P-glycoproteins, albeit at a low rate. PSC833 has a very high affinity for the MDR1 P-glycoprotein, and its Michaelis constant (Km) for transport is 50 nM, fourfold lower than for cyclosporin A. Inhibition of drug transport by PSC833 is approximately eightfold less effective in 100% fetal bovine serum than in tissue culture medium containing 10% serum. The concentration of PSC833 necessary to fully inhibit transport of digoxin and paclitaxel (Taxol) under complete (i.e., 100%) serum conditions is higher than the plasma concentrations achieved in clinical trials. CONCLUSIONS: Although PSC833 binds efficiently to the MDR1 P-glycoprotein and is released only sluggishly, the high concentrations of PSC833 necessary to inhibit this P-glycoprotein under complete serum conditions in our in vitro system suggest that it may be difficult for PSC833 alone to produce total inhibition of P-glycoprotein activity in patients.

Development and first use of a novel cylindrical ball bearing phantom for 9-DOF geometric calibrations of flat panel imaging devices used in image-guided ion beam therapy.

Image guidance during highly conformal radiotherapy requires accurate geometric calibration of the moving components of the imager. Due to limited manufacturing accuracy and gravity-induced flex, an x-ray imager's deviation from the nominal geometrical definition has to be corrected for. For this purpose a ball bearing phantom applicable for nine degrees of freedom (9-DOF) calibration of a novel cone-beam computed tomography (CBCT) scanner was designed and validated. In order to ensure accurate automated marker detection, as many uniformly distributed markers as possible should be used with a minimum projected inter-marker distance of 10 mm. Three different marker distributions on the phantom cylinder surface were simulated. First, a fixed number of markers are selected and their coordinates are randomly generated. Second, the quasi-random method is represented by setting a constraint on the marker distances in the projections. The third approach generates the ball coordinates helically based on the Golden ratio, ϕ. Projection images of the phantom incorporating the CBCT scanner's geometry were simulated and analysed with respect to uniform distribution and intra-marker distance. Based on the evaluations a phantom prototype was manufactured and validated by a series of flexmap calibration measurements and analyses. The simulation with randomly distributed markers as well as the quasi-random approach showed an insufficient uniformity of the distribution over the detector area. The best compromise between uniform distribution and a high packing fraction of balls is provided by the Golden section approach. A prototype was manufactured accordingly. The phantom was validated for 9-DOF geometric calibrations of the CBCT scanner with independently moveable source and detector arms. A novel flexmap calibration phantom intended for 9-DOF was developed. The ball bearing distribution based on the Golden section was found to be highly advantageous. The phantom showed satisfying results for calibrations of the CBCT scanner and provides the basis for further flexmap correction and reconstruction developments.

Impaired axonal regeneration in alpha7 integrin-deficient mice.

The interplay between growing axons and the extracellular substrate is pivotal for directing axonal outgrowth during development and regeneration. Here we show an important role for the neuronal cell adhesion molecule alpha7beta1 integrin during peripheral nerve regeneration. Axotomy led to a strong increase of this integrin on regenerating motor and sensory neurons, but not on the normally nonregenerating CNS neurons. alpha7 and beta1 subunits were present on the axons and their growth cones in the regenerating facial nerve. Transgenic deletion of the alpha7 subunit caused a significant reduction of axonal elongation. The associated delay in the reinnervation of the whiskerpad, a peripheral target of the facial motor neurons, points to an important role for this integrin in the successful execution of axonal regeneration.

Crystal structure of three consecutive laminin-type epidermal growth factor-like (LE) modules of laminin gamma1 chain harboring the nidogen binding site.

The structure of three consecutive laminin-type EGF-like (LE) modules of mouse laminin gammma1 chain, gamma1III3-5 (positions 738 to 899), has been determined by multiple isomorphous replacement in a crystal of space group p6(4)22 (a=b=74.57 angstroms, c = 185.11 angstroms and gamma = 120 degrees). The crystal structure was refined using restrained crystallographic refinement to an R-factor of 19.72% for 14,983 independent reflections with intensities F(obs)> 0 at 2.1 angstroms resolution, with root mean square deviation of 0.012 angstroms and 1.690 degrees from ideal bond lengths and bond angles, respectively. The final model consisted of 1179 (non-hydrogen) protein atoms within 162 residues and 119 water molecules. The molecule showed a rod-like structure of about 76 angstroms length with individual modules twisted relative to each other by about 70 degrees. Each module has the same disulfide bond connections Cys1-Cys3 (loop a), Cys2-Cys4 (loop b), Cys5-Cys6 (loop c) and Cys7-Cys8 (loop d), the first three being identical to epidermal growth factor (EGF). All three LE modules showed little secondary structure which was mainly restricted to loop d, but they differed in several other details of their structure. The interface contacts between the LE modules are based on hydrogen bonds and hydrophobic interactions between the hydrophobic core of loop d of the preceding module and the first cysteine and an exposed residue in loop b of the following module. Module 4 was previously shown to contribute the major nidogen binding site of laminis and site-directed mutagenesis demonstrated a specific binding role for Asp800, Asn802, Val804 and Tyr819 in loops a and c. The side-chain of these four residues are all located on the surface in a linear array and separated by a distance of 17 angstroms between Tyr819 and Val804. The entire nidogen binding site is stabilized via main-chain hydrogen bonds which are in part derived from the link between loops b and c (residues Leu815 and Lys816). The data demonstrate the unique nature of the LE modules and only a remote similarity to EGF. They also indicate that the crucial residues in the binding loops provide direct contacts with nidogen and explain the synergism between loops a and c which is essential for binding.

Changes in calcium and collagen IV binding caused by mutations in the EF hand and other domains of extracellular matrix protein BM-40 (SPARC, osteonectin).

Recombinant expression in a human kidney cell-line was used to prepare mutant human BM-40 with deletions including the N-terminal acidic domain, a central alpha-helical domain and the C-terminal EF hand domain. Two putative EF hand motifs were altered by point mutations. Only elimination of the whole EF hand domain or its single disulfide bond decreased production and secretion indicating that the C-terminal region of BM-40 is essential for correct folding. Deletions in the alpha-helical domain changed a single disulfide bond in this domain and caused oligomerization. Several mutations resulted in significant conformational changes as shown by CD spectroscopy and epitope analysis. Fluorescence titration demonstrated a single high-affinity (Kd = 0.08 microM) calcium-binding site with a low dissociation rate constant. A Glu to Lys mutation in the -Z position of the C-terminal EF hand motif and lack of a stabilizing disulfide bridge caused a 30 to 100-fold reduction in calcium affinity, while an Asp to Lys mutation in the X position had only a small effect. Deletions in the alpha-helical domain caused an even more dramatic reduction in calcium binding and abolished calcium-dependent binding of BM-40 to collagen IV. Both binding properties are critically dependent on a conformational interaction between the EF hand and the alpha-helical domain, which contains the collagen-binding site.

Structure of the nidogen binding LE module of the laminin gamma1 chain in solution.

The structure of the single LE module between residues 791 and 848 of the laminin gamma1 chain, which contains the high affinity binding site for nidogen, has been probed using NMR methods. The module folds into an autonomous domain which has a stable and unique three-dimensional (3D) structure in solution. The 3D structure was determined on the basis of 362 interproton distance constraints derived from nuclear Overhauser enhancement measurements and 39 phi angles, supplemented by 5 psi and 22 chi1 angles. The main features of the NMR structures are two-stranded antiparallel beta-sheets which are separated by loops and cross-connected by four disulfide bridges. The N-terminal segment which contains the first three disulfide bridges is similar to epidermal growth factor. The C-terminal segment has an S-like backbone profile with a crossover at the last disulfide bridge and comprises two three-residue long beta-strands that form an antiparallel beta-sheet. The LE module possesses an exposed nidogen binding loop that projects away from the main body of the protein. The side-chains of three amino acids which are crucial for binding (Asp, Asn, Val) are all exposed at the domain surface. An inactivating Asn-Ser mutation in this region showed the same 3D structure indicating that these three residues, and possibly an additional Tyr in an adjacent loop, provide direct contacts in the interaction with nidogen.

Roots of a social brain: developmental models of emerging animacy-detection mechanisms.

Here, we review evidence of unlearned predispositions to orient toward visual and auditory cues associated with the presence of animate creatures. We concentrate on studies on chicks of galliform species, whose behavioural preferences for social partners are analyzed in a comparative perspective with respect to the human developmental literature. The emerging nature of chicks' social predispositions is discussed in relation to the underlying physiological mechanisms and to the role of genetic and environmental factors in their development. In the second part of the review, we summarize evidence on the neural substrate of the animacy detectors, again focusing on our animal model of election, the domestic chick. On the basis of a substantial amount of indirect evidence, subpallial structures, among which the optic tectum (homologous to the mammalian superior colliculus), seem to comprise the most probable candidates. We also discuss some preliminary evidence of different brain activity, measured by IEG expression, in chicks exposed to predisposed or a non-predisposed stimulus.

Structural basis of beta 1 integrin-mediated cell adhesion to a large heparan sulfate proteoglycan from basement membranes.

In a cell attachment assay, several cell lines were found to adhere and spread on heparan sulfate proteoglycan purified from a basement membrane-producing tumor. This adhesion was clearly distinct from that on laminin. Cell adhesion to the proteoglycan was completely inhibited by three different antibodies against integrin beta 1 subunit, while inhibitory antibodies against beta 3 and alpha 2 to alpha 6 subunits were without strong effects. Removal of heparan sulfate from the proteoglycan diminished cell attachment, but addition of heparin to the cells did not affect adhesion to the proteoglycan. This suggests that both the heparan sulfate side chains and core protein structures are required for efficient cell adhesion. Studies with proteolytic fragments and synthetic RGD peptides showed that the single RGD sequence of mouse proteoglycan is not involved in cellular recognition. Characterization of fragments also allowed to localize cell adhesion and heparan sulfate attachment sites to the same 160 kDa core protein structure but not to fragments derived from the N-terminal portion of the proteoglycan.

Mutations in the pilz group genes disrupt the microtubule cytoskeleton and uncouple cell cycle progression from cell division in Arabidopsis embryo and endosperm.

Organised cell division and expansion play important roles in plant embryogenesis. To address their cellular basis, we have analysed Arabidopsis abnormal-embryo mutants which were isolated for their characteristic phenotype: mutant embryos are small, mushroom-shaped ("pilz") and consist of only one or few large cells each containing one or more variably enlarged nuclei and often cell wall stubs. These 23 mutants represent four genes, PFIFFERLING, HALLIMASCH, CHAMPIGNON, and PORCINO, which map to different chromosomes. All four genes have very similar mutant phenotypes although porcino embryos often consisted of only one large cell. The endosperm did not cellularise and contained a variably reduced number of highly enlarged nuclei. By contrast, genetic evidence suggests that these genes are not required for gametophyte development. Expression of cell cycle genes, Cdc2a, CyclinA2 and CyclinB1, and the cytokinesis-specific KNOLLE gene was not altered in mutant embryos. However, KNOLLE syntaxin accumulated in patches but no KNOLLE-positive structure resembling a forming cell plate occurred in mitotic cells. A general defect in microtubule assembly was observed in all mutants. Interphase cells lacked cortical microtubules, and spindles were absent from mitotic nuclei although in rare cases, short stubs of microtubules were attached to partially condensed chromosomes. Our results suggest that the cellular components affected by the pilz group mutations are necessary for continuous microtubule organisation, mitotic division and cytokinesis but do not mediate cell cycle progression.