The structure of cell wall alpha-glucan from fission yeast

Morphology and structural integrity of fungal cells depend on cell wall polysaccharides. The chemical structure and biosynthesis of two types of these polysaccharides, chitin and (1-->3)-beta-glucan, have been studied extensively, whereas little is known about alpha-glucan. Here we describe the chemical structure of alpha-glucan isolated from wild-type and mutant cell walls of the fission yeast Schizosaccharomyces pombe. Wild-type alpha-glucan was found to consist of a single population of linear glucose polymers, approximately 260 residues in length. These glucose polymers were composed of two interconnected linear chains, each consisting of approximately 120 (1-->3)-linked alpha-d-glucose residues and some (1-->4)-linked alpha-D-glucose residues at the reducing end. By contrast, alpha-glucan of an alpha-glucan synthase mutant with an aberrant cell morphology and reduced alpha-glucan levels consisted of a single chain only. We propose that alpha-glucan biosynthesis involves an ordered series of events, whereby two alpha-glucan chains are coupled to create mature cell wall alpha-glucan. This mature form of cell wall alpha-glucan is essential for fission-yeast morphogenesis.     

Sonic hedgehog regulates otic capsule chondrogenesis and inner ear development in the mouse embryo

Development of the cartilaginous capsule of the inner ear is dependent on interactions between otic epithelium and its surrounding periotic mesenchyme. During these tissue interactions, factors endogenous to the otic epithelium influence the differentiation of the underlying periotic mesenchyme to form a chondrified otic capsule. We report the localization of Sonic hedgehog (Shh) protein and expression of the Shh gene in the tissues of the developing mouse inner ear. We demonstrate in cultures of periotic mesenchyme that Shh alone cannot initiate otic capsule chondrogenesis. However, when Shh is added to cultured periotic mesenchyme either in combination with otic epithelium or otic epithelial-derived fibroblast growth factor (FGF2), a significant enhancement of chondrogenesis occurs. Addition of Shh antisense oligonucleotide (AS) to cultured periotic mesenchyme with added otic epithelium decreases levels of endogenous Shh and suppresses the chondrogenic response of the mesenchyme cells, while supplementation of Shh AS-treated cultures with Shh rescues cultures from chondrogenic inhibition. We demonstrate that inactivation of Shh by targeted mutation produces anomalies in the developing inner ear and its surrounding capsule. Our results support a role for Shh as a regulator of otic capsule formation and inner ear development during mammalian embryogenesis.     

FIP-2, an IkappaB-kinase-gamma-related protein,is associated with the Golgi apparatus and translocates to the marginal band during chicken erythroblast differentiation

Using an antiserum directed against marginal band associated proteins of chicken erythrocytes we isolated clones encoding the chicken homolog of 14.7K-interacting protein 2 (FIP-2), a protein potentially involved in tumor necrosis factor-alpha/nuclear factor-kappaB signaling, from a chicken erythroblast cDNA library. We found that chicken FIP-2 was expressed in a variety of tissues and cell types, but unlike its human counterpart, alternative splicing does not appear to take place. Analysis of intracellular localization revealed that FIP-2 was concentrated at the Golgi apparatus in most cells. Perturbation of the Golgi structure without loss of Golgi function (by treatment with nocodazole) resulted in a retention of FIP-2 at the dispersed Golgi fragments. In contrast, disruption of both Golgi structure and function (by brefeldin A) led to a loss of FIP-2 from Golgi membranes. Remarkably, during erythroblast differentiation FIP-2 was found to translocate from the Golgi to the marginal band. Our results support the hypothesis of a function of the Golgi apparatus in signal transduction. Moreover, our results raise the possibility that the marginal band of chicken erythrocytes, in addition to its role in morphogenesis, has a function in signal transduction and that FIP-2 is in some way involved in its formation.     

Dendrite-like process formation and cytoskeletal remodeling regulated by delta-catenin expression

Actin- and microtubule-mediated changes in cell shape are essential for many cellular activities. However, the molecular mechanisms underlying the interplay between the two are complex and remain obscure. Here we show that the expression of delta-catenin (or NPRAP/Neurojungin), a member of p120(ctn) subfamily of armadillo proteins can induce the branching of dendrite-like processes in 3T3 cells and enhance dendritic morphogenesis in primary hippocampal neurons. This induction of branching phenotype involves initially the disruption of filamentous actin, and requires the growth of microtubules. The carboxyl-terminal truncation mutant of delta-catenin can cluster and redistribute the full-length protein, and dominantly inhibit its branching effect. delta-Catenin forms protein complexes and can bind directly to actin in vitro. The carboxyl-terminal truncation of delta-catenin does not interfere with its actin-binding capability; therefore the actin interaction alone is not sufficient for the induction of dendrite-like processes. When delta-catenin-transformed cells establish elaborate dendrite-like branches, the main cellular processes become stabilized and resist the disruption of both actin filaments and microtubules, as determined by fluorescent light microscopy and time-lapse recording analyses. We suggest that delta-catenin can effect a biphasic cytoskeletal remodeling event which differentially regulates actin and microtubules and promotes cellular morphogenesis.     

Slow local movements of collagen fibers by fibroblasts drive the rapid global self-organization of collagen gels

Aclassic model for tissue morphogenesis is the formation of ligament-like straps between explants of fibroblasts placed in collagen gels. The patterns arise from mechanical forces exerted by cells on their substrates (Harris et al., 1981). However, where do such straps come from, and how are slow local movements of cells transduced into dramatic long-distance redistributions of collagen? We embedded primary mouse skin and human periodontal ligament fibroblasts in collagen gels and measured the time course of patterning by using a novel computer algorithm to calculate anisotropy, and by tracking glass beads dispersed in the gel. As fibroblasts began to spread into their immediate environments, a coordinated rearrangement of collagen commenced throughout the gel, producing a strap on a time scale of minutes. Killing of cells afterwards resulted in a partial relaxation of the matrix strain. Surprisingly, relatively small movements of collagen molecules on the tensile axis between two pulling explants induced a much larger concomitant compression of the gel perpendicular to the axis, organizing and aligning fibers into a strap. We propose that this amplification is due to the geometry of the collagen matrix, and that analogous amplified movements may drive morphological changes in other biological meshes, both outside and inside the cell.     

Essential role of growth factor receptor-mediated signal transduction through the mitogen-activated protein kinase pathway in early embryogenesis of the echinoderm

In this study it was shown that growth factor receptors (GFR) play a crucial role in early embryogenesis of the echinoderms Hemicentrotus pulcherrimus and Clypeaster japonicus by transmitting signals to the mitogen-activated protein kinase (MAPK) pathway. The phosphorylation ratio of extracellular signal-regulated kinase 1 (ERK1) changed dynamically during early embryogenesis and showed a peak at the swimming blastula (sBl) stage. Suramin, an inhibitor of GFR, when applied during the sBl stage perturbed morphogenesis, including primary mesenchyme cell (PMC) migration, cell proliferation, archenteron elongation, spiculogenesis, pigment cell differentiation and phosphorylation of myosin light chains (MLC). Genistein, a receptor-type protein tyrosine kinase inhibitor, severely inhibited PMC migration, gastrulation and the phosphorylation of MLC. Manumycin A, a Ras inhibitor, inhibited spiculogenesis and invagination. PD98059, a MAPK/ERK kinase inhibitor, perturbed early PMC migration and pigment cell differentiation, but not spiculogenesis and gastrulation (although these two events were significantly delayed). PMC ingression was not perturbed by genistein, suramin, manumycin A or PD98059. All of the inhibitors perturbed the phosphorylation of ERK1, which was completely restored by exogenous platelet-derived growth factor (PDGF)-AB. PDGF-AB also partially restored elongation of the archenteron by restoring cell proliferation that had been perturbed by suramin.     

The role of GDNF in patterning the excretory system

Mesenchymal-epithelial interactions are an important source of information for pattern formation during organogenesis. In the developing excretory system, one of the secreted mesenchymal factors thought to play a critical role in patterning the growth and branching of the epithelial ureteric bud is GDNF. We have tested the requirement for GDNF as a paracrine chemoattractive factor by altering its site of expression during excretory system development. Normally, GDNF is secreted by the metanephric mesenchyme and acts via receptors on the Wolffian duct and ureteric bud epithelium. Misexpression of GDNF in the Wolffian duct and ureteric buds resulted in formation of multiple, ectopic buds, which branched independently of the metanephric mesenchyme. This confirmed the ability of GDNF to induce ureter outgrowth and epithelial branching in vivo. However, in mutant mice lacking endogenous GDNF, kidney development was rescued to a substantial degree by GDNF supplied only by the Wolffian duct and ureteric bud. These results indicate that mesenchymal GDNF is not required as a chemoattractive factor to pattern the growth of the ureteric bud within the developing kidney, and that any positional information provided by the mesenchymal expression of GDNF may provide for renal branching morphogenesis is redundant with other signals.     

Genetic engineering in floriculture

The global flower industry thrives on novelty. Genetic engineering is providing a valuable means of expanding the floriculture gene pool so promoting the generation of new commercial varieties. Commercialisation of genetically engineered flowers is currently confined to novel coloured carnations. However, further products are expected given the level of activity in the field. In general terms engineered traits are valuable to either the consumer or the producer. At present only consumer traits appear able to provide a return capable of supporting what is still a relatively expensive molecular breeding tool. The biosynthesis of floral pigments, particularly anthocyanins, has been elucidated in great detail in model flowers such as petunia. This knowledge is now being applied to an understanding of a wide range of other flowers and providing a means of targeting colour modification in these species. The engineering of novel traits in a given variety also rests on capabilities in plant transformation that are continuing to expand at a rapid rate. The expression of genes transferred across genera is not always predictable and so requires considerable trial and error to arrive at stable phenotype of commercial interest. Manipulation of metabolic pathways, often requiring introduction of multiple genes can also be problematic. This is a reflection of the complexity of interactions within and between cells at a gene and gene product level. An understanding of gene function is only an essential first step in engineering novel traits. The production of novel flower colour has been the first success story in floriculture genetic engineering. Other traits that have received attention include floral scent, floral and plant morphology, senescence of flowers both on the plant and post-harvest and disease resistance.     

Virus factories: associations of cell organelles for viral replication and morphogenesis

Genome replication and assembly of viruses often takes place in specific intracellular compartments where viral components concentrate, thereby increasing the efficiency of the processes. For a number of viruses the formation of 'factories' has been described, which consist of perinuclear or cytoplasmic foci that mostly exclude host proteins and organelles but recruit specific cell organelles, building a unique structure. The formation of the viral factory involves a number of complex interactions and signalling events between viral and cell factors. Mitochondria, cytoplasmic membranes and cytoskeletal components frequently participate in the formation of viral factories, supplying basic and common needs for key steps in the viral replication cycle.