Spatial and temporal gene expression patterns occur during corm development.

We investigated gene expression patterns that occur during taro corm development. Two-dimensional gel electrophoresis identified several different prevalent proteins that accumulate during corm development. Microsequencing studies indicated that some of these proteins are related to taste-modifying proteins, such as curculin and miraculin, and proteins found in other storage organs, such as sporamin and the Kunitz trypsin inhibitor. A curculin-encoding cDNA clone, designated as TC1, was identified that corresponds to a highly prevalent 1-kb corm mRNA. The TC1 mRNA accumulates during corm development, is more prevalent in corm apical than basal regions, and is either absent, or present at low concentrations, in other vegetative organs such as the leaf and root. In situ hybridization experiments showed that the TC1 mRNA is highly concentrated in corm storage parenchyma cells and is absent, or present in reduced concentrations, in other corm cells and tissues. Our results show that corm development is associated with the differentiation of specialized cells and tissues, and that these differentiation events are coupled with the temporal and spatial expression of corm-specific genes.     

Modelling microtubule patterns

The cellular cytoskeleton is well studied in terms of its biological and physical properties, making it an attractive subject for systems approaches. Here, we describe the experimental and theoretical strategies used to study the collective behaviour of microtubules and motors. We illustrate how this led to the beginning of an understanding of dynamic cellular patterns that have precise functions.     

Microtubule oscillations. Role of nucleation and microtubule number concentration

Microtubules are capable of performing synchronized oscillations of assembly and disassembly which has been explained by reaction mechanisms involving tubulin subunits, oligomers, microtubules, and GTP. Here we address the question of how microtubule nucleation or their number concentration affects the oscillations. Assembly itself requires a critical protein concentration (Cc), but oscillations require in addition a critical microtubule number concentration (CMT). In spontaneous assembly this can be achieved with protein concentrations Cos well above the critical concentration Cc because this enhances the efficiency of nucleation. Seeding with microtubules can either generate oscillations or suppress them, depending on how the seeds alter the effective microtubule number concentration. The relative influence of microtubule number and total protein concentrations can be varied by the rate at which assembly conditions are induced (e.g. by a temperature rise): Fast T-jumps induce oscillations because of efficient nucleation, slow ones do not. Oscillations become damped for several reasons. One is the consumption of GTP, the second is a decrease in microtubule number, and the third is that the ratio of microtubules in the two phases (growth-competent and shrinkage-competent) approach a steady state value. This ratio can be perturbed, and the oscillations restarted, by a cold shock, addition of seeds, addition of GTP, or fragmentation. Each of these is equivalent to a change in the effective microtubule number concentration.     

Comparison of the four mouse fasciclin-containing genes expression patterns during valvuloseptal morphogenesis

All four mammalian fasciclin-containing genes are expressed in the adult valves and are localized in partially overlapping and reciprocal patterns during cardiovascular development. Spatiotemporal comparison of the fasciclin-containing secreted adhesion genes, TGFbeta induced clone H3 (betaigH3) and periostin, revealed that they are co-localized within the outflow tract endocardial cushions, but that betaigH3 expression is restricted to the septal cushions within the atrioventricular canal. Conversely, the fasciclin-containing transmembrane gene, stabilin-1, is predominately expressed in the endocardial layer overlaying the cushions and lining the developing heart. However, expression of the fasciclin-containing transmembrane gene, stabilin-2 is only present in the post-natal mature valve endothelial cells. These data illustrate for the first time that the primitive endocardial cushions dynamically express multiple fasciclin-containing adhesion molecules as they undergo the key steps of seeding, proliferation, differentiation, fusion, mesenchymal condensation and remodeling during mouse heart development.     

Dynamic patterns of subcellular protein localization during spore coat morphogenesis in Bacillus subtilis

Endospores of Bacillus subtilis are encased in a thick, proteinaceous shell known as the coat, which is composed of a large number of different proteins. Here we report the identification of three previously uncharacterized coat-associated proteins, YabP, YheD, and YutH, and their patterns of subcellular localization during the process of sporulation, obtained by using fusions of the proteins to the green fluorescent protein (GFP). YabP-GFP was found to form both a shell and a ring around the center of the forespore across the short axis of the sporangium. YheD-GFP, in contrast, formed two rings around the forespore that were offset from its midpoint, before it eventually redistributed to form a shell around the developing spore. Finally, YutH-GFP initially localized to a focus at one end of the forespore, which then underwent transformation into a ring that was located adjacent to the forespore. Next, the ring became a cap at the mother cell pole of the forespore that eventually spread around the entire developing spore. Thus, each protein exhibited its own distinct pattern of subcellular localization during the course of coat morphogenesis. We concluded that spore coat assembly is a dynamic process involving diverse patterns of protein assembly and localization.     

Bud morphogenesis and the actin and microtubule cytoskeletons during budding in the corn smut fungus,Ustilago maydis

Ustilago maydis is a dimorphic Basidiomycete fungus with a yeast-like form and a hyphal form. Here we present a comprehensive analysis of bud formation and the actin and microtubule cytoskeletons of the yeast-like form during the cell cycle. We show that bud morphogenesis entails a series of shape changes, initially a tubular or conical structure, culminating in a cigar-shaped cell connected to the mother cell by a narrow neck. Labelling of cells with concanavalin A demonstrated that growth occurs at bud tip. Indirect immunofluorescence studies revealed that the actin cytoskeleton consists of patches and cables that polarize to the presumptive bud site and the bud tip and an actin ring that forms at the neck region. Because the bud tip corresponds to the site of active cell wall growth, we hypothesize that actin is involved in secretion of cell wall components. The microtubule cytoskeleton has recently been shown to consist of a cytoplasmic network during interphase that disassembles at mitosis when a spindle and astral microtubules are formed. We have carried out studies of U. maydis cells synchronized by the microtubule-depolymerizing drug thiabendazole which allow us to construct a temporal sequence of steps in spindle formation and spindle elongation during the cell cycle. These studies suggest that astral microtubules may be involved in early stages of spindle orientation and migration of the nucleus into the bud and that the spindle pole bodies may be involved in reestablishment of the cytoplasmic microtubule network.     

Regulation of cellular differentiation during Dictyostelium morphogenesis.

In Dictyostelium there are multiple prestalk cell types that have a complex pattern of directed cell movement during slug formation and culmination. Three extracellular signals, cyclic AMP, DIF and ammonia, control cell type differentiation. Recently there has been considerable progress in understanding their modes of action and interaction.     

Desmin expression during early mouse tongue morphogenesis.

Occipital somites provide progenitor cells for craniofacial muscle development including the tongue musculature. Serum-derived factors are assumed to be pre-requisite for myogenesis in vitro. To test these assertions, we designed experiments to determine whether early mouse tongue development in general, and desmin localization in particular, were expressed during the development of embryonic mouse first branchial arch explants cultured in serumless, chemically-defined medium. Immunohistochemical techniques determined the chronology and positions of desmin expression during early craniofacial development. Occipital somites expressed desmin at E9 (9 days +/- 2 h post-fertilization, 18-20 somites). A discrete cell migration pathway initiating in the somites and terminating in the lateral lingual processes of the tongue primordium was defined based upon desmin expression patterns in E9-E11 embryos and computer-assisted three dimensional reconstructions. The in vitro model system was permissive for tongue morphogenesis, allowing development and fusion of the lateral lingual processes with the tuberculum impar. During culture myoblasts were not observed to fuse into myotubes with sarcomeric assembly, even though explant myoblasts produced muscle-specific protein. E10 explants cultured for 9 days demonstrated a five-fold increase in cell number that expressed desmin (P less than 0.05) when compared to the E10 starting material. We interpret these results to indicate that the tongue myogenic cell lineage was determined between E8 and E11, and that this resident population expanded within explants cultured in serumless medium by several explanations: (i) cells other than progenitor myoblasts (e.g., satellite cells) were induced to become myoblasts, and/or (ii) progenitor myoblasts within the original explants expanded by cell division in the absence of serum factors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of Sindbis virus glycoproteins during morphogenesis.

In cells infected with the Sindbis temperature-sensitive mutants ts-23 and ts-10 (complementation group D), which contain a defect in the envelope glycoprotein E1, the precursor polypeptide PE2 is not cleaved to the envelope glycoprotein E2 at the nonpermissive temperature. This defect is phenotypically identical to the defect observed in the complementation group E mutant, ts-20. The lesion in ts-23 is reversible upon shift to permissive temperature, whereas that of ts-10 is not. Antiserum against whole virus, E1, or E2 also prevents the cleavage of PE2 in cells infected with wild-type Sindbis virus. Because the cleavage of PE2 is inhibited by the lesion in mutants that are genotypically distinct and by anti-E1 or -E2 serum, it appears that PE2 and E1 exist as a complex in the membrane of the infected cell.     

Lipid synthesis during morphogenesis of Mucor racemosus.

Lipid synthesis increases coordinately with protein and RNA synthesis during morphogenesis of Mucor racemosus. The lipid synthesis inhibitor cerulenin can completely block morphogenesis under conditions in which cell growth continues. An increase in phospholipid turnover may be an important correlate to morphogenesis of Mucor spp., especially the turnover of phosphotidyl inositol and phosphatidyl ethanolamine. The increase in ornithine decarboxylase, which occurs during morphogenesis, is inhibited by the addition of cerulenin.     

GTP hydrolysis during microtubule assembly

The GTP cap model of dynamic instability [Mitchison, T., & Kirschner, M.W. (1984) Nature (London) 312, 237] postulates that a GTP cap at the end of most microtubules stabilizes the polymer and allows continuing assembly of GTP-tubulin subunits while microtubules without a cap rapidly disassemble. This attractive explanation for observed microtubule behavior is based on the suggestion that hydrolysis of GTP is not coupled to assembly but rather takes place as a first-order reaction after a subunit is assembled onto a polymer end. Carlier and Pantaloni [Carlier, M., & Pantaloni, D. (1981) Biochemistry 20, 1918] reported a lag of hydrolysis behind microtubule assembly and a first-order rate constant for hydrolysis (kh) of 0.25/min. A lag has not been demonstrated by other investigators, and a kh value that specifies such a slow rate of hydrolysis is difficult to reconcile with reported steady-state microtubule growth rates and frequencies of disassembly. We have looked for a lag using tubulin free of microtubule-associated protein at concentrations of 18.5-74 microM, assembly with and without glycerol, and two independent assays of GTP hydrolysis. No lag was observed under any of the conditions employed, with initial rates of hydrolysis increasing in proportion to rates of assembly. If hydrolysis is uncoupled from assembly, we estimate that kh must be at least 2.5/min and could be much greater, a result that we argue may be advantageous to the GTP cap model. We also describe a preliminary model of assembly coupled to hydrolysis that specifies formation and loss of a GTP cap, thus allowing dynamic instability.     

Bud6 directs sequential microtubule interactions with the bud tip and bud neck during spindle morphogenesis in Saccharomyces cerevisiae

In budding yeast, spindle polarity relies on a precise temporal program of cytoplasmic microtubule-cortex interactions throughout spindle assembly. Loss of Clb5-dependent kinase activity under conditions of attenuated Cdc28 function disrupts this program, resulting in diploid-specific lethality. Here we show that polarity loss is tolerated by haploids due to a more prominent contribution of microtubule-neck interactions to spindle orientation inherent to haploids. These differences are mediated by the relative partition of Bud6 between the bud tip and bud neck, distinguishing haploids from diploids. Bud6 localizes initially to the bud tip and accumulates at the neck concomitant with spindle assembly. bud6Delta mutant phenotypes are consistent with Bud6's role as a cortical cue for cytoplasmic microtubule capture. Moreover, mutations that affect Bud6 localization and partitioning disrupt the sequential program of microtubule-cortex interactions accordingly. These data support a model whereby Bud6 sequentially cues microtubule capture events at the bud tip followed by capture events at the bud neck, necessary for correct spindle morphogenesis and polarity.     

Dephosphorylation of tubulin-bound guanosine triphosphate during microtubule assembly.

1. Tubulin purified from porcine brain in the presence of GTP contained 0.16 mole of GDP and 0.73 mole of GTP per 60,000 g of protein. 2. Microtubules reconstituted from the purified tubulin contained 0.43 mole of GDP and 0.41 mole of GTP per 60,000 g of protein. Guanine nucleotide bound to the exchangeable site of tubulin was converted to GDP during microtubule assembly, while GTP at the non-exchangeable site remained intact. 3. Guanine nucleotide which had been bound to the exchangeable site of tubulin before microtubule assembly was also exchangeable during disassembly.     

Detecting conserved interaction patterns in biological networks.

Molecular interaction data plays an important role in understanding biological processes at a modular level by providing a framework for understanding cellular organization, functional hierarchy, and evolutionary conservation. As the quality and quantity of network and interaction data increases rapidly, the problem of effectively analyzing this data becomes significant. Graph theoretic formalisms, commonly used for these analysis tasks, often lead to computationally hard problems due to their relation to subgraph isomorphism. This paper presents an innovative new algorithm, MULE, for detecting frequently occurring patterns and modules in biological networks. Using an innovative graph simplification technique based on ortholog contraction, which is ideally suited to biological networks, our algorithm renders these problems computationally tractable and scalable to large numbers of networks. We show, experimentally, that our algorithm can extract frequently occurring patterns in metabolic pathways and protein interaction networks from the KEGG, DIP, and BIND databases within seconds. When compared to existing approaches, our graph simplification technique can be viewed either as a pruning heuristic, or a closely related, but computationally simpler task. When used as a pruning heuristic, we show that our technique reduces effective graph sizes significantly, accelerating existing techniques by several orders of magnitude! Indeed, for most of the test cases, existing techniques could not even be applied without our pruning step. When used as a stand-alone analysis technique, MULE is shown to convey significant biological insights at near-interactive rates. The software, sample input graphs, and detailed results for comprehensive analysis of nine eukaryotic PPI networks are available at www.cs.purdue.edu/homes/koyuturk/mule.     

Microtubule dynamics during infection-related morphogenesis of Colletotrichum lagenarium.

Using a green fluorescent protein (GFP)-tubulin fusion protein, we have investigated the dynamic rearrangement of microtubules during appressorium formation of Colletotrichum lagenarium. Two alpha-tubulin genes of C. lagenarium were isolated, and GFP-alpha-tubulin protein was expressed in this fungus. The strain expressing the fusion protein formed fluorescent filaments that were disrupted by a microtubule-depolymerizing drug, benomyl, demonstrating successful visualization of microtubules. In preincubated conidia, GFP-labeled interphase microtubules, showing random orientation, were observed. At conidial germination, microtubules oriented toward a germination site. At nuclear division, when germ tubes had formed appressoria, mitotic spindles appeared inside conidia followed by disassembly of interphase microtubules. Remarkably, time-lapse views showed that interphase microtubules contact a microtubule-associated center at the cell cortex of conidia that is different from a nuclear spindle pole body (SPB) before their disassembly. Duplicated nuclear SPBs separately moved toward conidium and appressorium accompanied by astral microtubule formation. Benomyl treatment caused movement of both daughter nuclei into 70% of appressoria and affected appressorium morphogenesis. In conidia elongating hyphae without appressoria, microtubules showed polar elongation which is distinct from their random orientation inside appressoria.     

Distinct ECM mechanosensing pathways regulate microtubule dynamics to control endothelial cell branching morphogenesis

During angiogenesis, cytoskeletal dynamics that mediate endothelial cell branching morphogenesis during vascular guidance are thought to be regulated by physical attributes of the extracellular matrix (ECM) in a process termed mechanosensing. Here, we tested the involvement of microtubules in linking mechanosensing to endothelial cell branching morphogenesis. We used a recently developed microtubule plus end-tracking program to show that specific parameters of microtubule assembly dynamics, growth speed and growth persistence, are globally and regionally modified by, and contribute to, ECM mechanosensing. We demonstrated that engagement of compliant two-dimensional or three-dimensional ECMs induces local differences in microtubule growth speed that require myosin II contractility. Finally, we found that microtubule growth persistence is modulated by myosin II-mediated compliance mechanosensing when cells are cultured on two-dimensional ECMs, whereas three-dimensional ECM engagement makes microtubule growth persistence insensitive to changes in ECM compliance. Thus, compliance and dimensionality ECM mechanosensing pathways independently regulate specific and distinct microtubule dynamics parameters in endothelial cells to guide branching morphogenesis in physically complex ECMs.