Mother Cell Side Research Articles

Spatio-temporal control of asymmetric septum positioning during sporulation in Bacillus subtilis

During sporulation, Bacillus subtilis forms an asymmetric septum, dividing the cell into two compartments, a mother cell and a forespore. The site of asymmetric septation is linked to the membrane where FtsZ and SpoIIE initiate the formation of the Z-ring and the E-ring, respectively. These rings then serve as a scaffold for the other cell division and peptidoglycan synthesizing proteins needed to build the septum. However, despite decades of research, not enough is known about how the asymmetric septation site is determined. Here, we identified and characterized the interaction between SpoIIE and RefZ. We show that these two proteins transiently co-localize during the early stages of asymmetric septum formation when RefZ localizes primarily from the mother cell side of the septum. We propose that these proteins and their interplay with the spatial organization of the chromosome play a role in controlling asymmetric septum positioning.

Asymmetric localization of the cell division machinery during Bacillus subtilis sporulation.

The Gram-positive bacterium Bacillus subtilis can divide via two modes. During vegetative growth, the division septum is formed at the midcell to produce two equal daughter cells. However, during sporulation, the division septum is formed closer to one pole to yield a smaller forespore and a larger mother cell. Using cryo-electron tomography, genetics and fluorescence microscopy, we found that the organization of the division machinery is different in the two septa. While FtsAZ filaments, the major orchestrators of bacterial cell division, are present uniformly around the leading edge of the invaginating vegetative septa, they are only present on the mother cell side of the invaginating sporulation septa. We provide evidence suggesting that the different distribution and number of FtsAZ filaments impact septal thickness, causing vegetative septa to be thicker than sporulation septa already during constriction. Finally, we show that a sporulation-specific protein, SpoIIE, regulates asymmetric divisome localization and septal thickness during sporulation.

GerM is required to assemble the basal platform of the SpoIIIA-SpoIIQ transenvelope complex during sporulation in Bacillus subtilis.

Sporulating Bacillus subtilis cells assemble a multimeric membrane complex connecting the mother cell and developing spore that is required to maintain forespore differentiation. An early step in the assembly of this transenvelope complex (called the A-Q complex) is an interaction between the extracellular domains of the forespore membrane protein SpoIIQ and the mother cell membrane protein SpoIIIAH. This interaction provides a platform onto which the remaining components of the complex assemble and also functions as an anchor for cell-cell signalling and morphogenetic proteins involved in spore development. SpoIIQ is required to recruit SpoIIIAH to the sporulation septum on the mother cell side; however, the mechanism by which SpoIIQ specifically localizes to the septal membranes on the forespore side has remained enigmatic. Here, we identify GerM, a lipoprotein previously implicated in spore germination, as the missing factor required for SpoIIQ localization. Our data indicate that GerM and SpoIIIAH, derived from the mother cell, and SpoIIQ, from the forespore, have reciprocal localization dependencies suggesting they constitute a tripartite platform for the assembly of the A-Q complex and a hub for the localization of mother cell and forespore proteins.

Peptidoglycan hydrolysis is required for assembly and activity of the transenvelope secretion complex during sporulation in Bacillus subtilis

Sporulating Bacillus subtilis cells assemble a transenvelope secretion complex that connects the mother cell and developing spore. The forespore protein SpoIIQ and the mother-cell protein SpoIIIAH interact across the double membrane septum and are thought to assemble into a channel that serves as the basement layer of this specialized secretion system. SpoIIQ is absolutely required to recruit SpoIIIAH to the sporulation septum on the mother-cell side, however the mechanism by which SpoIIQ is localized has been unclear. Here, we show that SpoIIQ localization requires its partner protein SpoIIIAH and degradation of the septal peptidoglycan (PG) by the two cell wall hydrolases SpoIID and SpoIIP. Our data suggest that PG degradation enables a second mother-cell-produced protein to interact with SpoIIQ. Cells in which both mother-cell anchoring mechanisms have been disabled have a synergistic sporulation defect suggesting that both localization factors function in the secretion complex. Finally, we show that septal PG degradation is critical for the assembly of an active complex. Altogether, these results suggest that the specialized secretion system that links the mother cell and forespore has a complexity approaching those found in Gram-negative bacteria and reveal that the sporulating cell must overcome similar challenges in assembling a transenvelope complex.

Subcellular localization of a sporulation membrane protein is achieved through a network of interactions along and across the septum

During the process of spore formation in Bacillus subtilis many membrane proteins localize to the sporulation septum where they play key roles in morphogenesis and cell-cell signalling. However, the mechanism by which these proteins are anchored at this site is not understood. In this report we have defined the localization requirements for the mother-cell membrane protein SpoIVFA, which anchors a signalling complex in the septal membrane on the mother cell side. We have identified five proteins (SpoIID, SpoIIP, SpoIIM, BofA and SpoIIIAH) synthesized in the mother cell under the control of sigma(E) and one protein (SpoIIQ) synthesized in the forespore under the control of sigma(F) that are all required for the proper localization of SpoIVFA. Surprisingly, these proteins appear to have complementary and overlapping anchoring roles suggesting that SpoIVFA is localized in the septal membrane through a web of protein interactions. Furthermore, we demonstrate a direct biochemical interaction between the extracellular domains of two of the proteins required to anchor SpoIVFA: the forespore protein SpoIIQ and the mother-cell protein SpoIIIAH. This result supports the idea that the web of interactions that anchors SpoIVFA is itself held in the septal membrane through a zipper-like interaction across the sporulation septum. Importantly, our results suggest that a second mechanism independent of forespore proteins participates in anchoring SpoIVFA. Finally, we show that the dynamic localization of SpoIIQ in the forespore is impaired in the absence of SpoIVFA but not SpoIIIAH. Thus, a complex web of interactions among mother cell and forespore proteins is responsible for static and dynamic protein localization in both compartments of the sporangium. We envision that this proposed network is involved in anchoring other sporulation proteins in the septum and that protein networks with overlapping anchoring capacity is a feature of protein localization in all bacteria.

SUMO Modification of Septin-interacting Proteins in Candida albicans

The initiation of bud and hyphal growth in the opportunistic fungal pathogen Candida albicans both involve polarized morphogenesis. However, there are many differences including the function of the septin proteins, a family of proteins involved in membrane organization in a wide range of organisms. Septins form a characteristic ring on the inner surface of the plasma membrane at the bud neck, whereas the septins are diffusely localized across emerging hyphal tips. In addition, septin rings are maintained at sites of septum formation in hyphae rather than being disassembled immediately after cytokinesis. The possibility that C. albicans septins are regulated by the small ubiquitin-like protein SUMO was examined in this study because the Saccharomyces cerevisiae septins were shown previously to be modified by SUMO (Smt3p). However, SUMO conjugation to septins was not detected during budding or hyphal morphogenesis in C. albicans. These results are supported by the lack of conserved SUMO consensus motifs between septins from the two organisms even after adjusting the predicted Cdc3p and Cdc12p septin sequences to account for mRNA splicing in C. albicans. Interestingly, a homolog of the Smt3p SUMO was identified in the C. albicans genome, and an epitope tagged version of Smt3p was conjugated to a variety of proteins. Immunofluorescence analysis showed prominent Smt3p SUMO localization at bud necks and sites of septum formation in hyphae similar to the septins. However, Smt3p was primarily detected on the mother cell side of the septin ring. A subset of these Smt3p-modified proteins co-immunoprecipitated with the septin Cdc11p. These results indicate that septin-associated proteins and not the septins themselves are the key target of SUMO modification at the bud neck in C. albicans.

Cell cycle-regulated attachment of the ubiquitin-related protein SUMO to the yeast septins.

SUMO is a ubiquitin-related protein that functions as a posttranslational modification on other proteins. SUMO conjugation is essential for viability in Saccharomyces cerevisiae and is required for entry into mitosis. We have found that SUMO is attached to the septins Cdc3, Cdc11, and Shs1/Sep7 specifically during mitosis, with conjugates appearing shortly before anaphase onset and disappearing abruptly at cytokinesis. Septins are components of a belt of 10-nm filaments encircling the yeast bud neck. Intriguingly, only septins on the mother cell side of the bud neck are sumoylated. We have identified four major SUMO attachment-site lysine residues in Cdc3, one in Cdc11, and two in Shs1, all within the consensus sequence (IVL)KX(ED). Mutating these sites eliminated the vast majority of bud neck-associated SUMO, as well as the bulk of total SUMO conjugates in G(2)/M-arrested cells, indicating that sumoylated septins are the most abundant SUMO conjugates at this point in the cell cycle. This mutant has a striking defect in disassembly of septin rings, resulting in accumulation of septin rings marking previous division sites. Thus, SUMO conjugation plays a role in regulating septin ring dynamics during the cell cycle.

The "pro" sequence of the sporulation-specific sigma transcription factor sigma(E) directs it to the mother cell side of the sporulation septum.

sigma(E), a mother cell-specific transcription factor of sporulating Bacillus subtilis, is derived from an inactive precursor protein (pro-sigma(E)). Activation of sigma(E) occurs when a sporulation-specific protease (SpoIIGA) cleaves 27 amino acids from the pro-sigma(E) amino terminus. This reaction is believed to take place at the mother cell-forespore septum. Using a chimera of pro-sigma(E) and green fluorescent protein (GFP) to visualize the intracellular location of pro-sigma(E) by fluorescence microscopy, and lysozyme treatment to separate the mother cell and forespore compartments, we determined that the pro-sigma(E)::GFP signal, localized to the forespore septum prior to lysozyme treatment, is restricted to the mother cell compartment after treatment. Thus, pro-sigma(E)::GFP had been sequestered to the mother cell side of the septum. This segregation of pro-sigma(E)::GFP, and presumably pro-sigma(E), to the mother cell is likely to be the reason why sigma(E) activity is restricted to that compartment.

A septin-based hierarchy of proteins required for localized deposition of chitin in the Saccharomyces cerevisiae cell wall.

Just before bud emergence, a Saccharomyces cerevisiae cell forms a ring of chitin in its cell wall; this ring remains at the base of the bud as the bud grows and ultimately forms part of the bud scar marking the division site on the mother cell. The chitin ring seems to be formed largely or entirely by chitin synthase III, one of the three known chitin synthases in S. cerevisiae. The chitin ring does not form normally in temperature-sensitive mutants defective in any of four septins, a family of proteins that are constituents of the "neck filaments" that lie immediately subjacent to the plasma membrane in the mother-bud neck. In addition, a synthetic-lethal interaction was found between cdc12-5, a temperature-sensitive septin mutation, and a mutant allele of CHS4, which encodes an activator of chitin synthase III. Two-hybrid analysis revealed no direct interaction between the septins and Chs4p but identified a novel gene, BNI4, whose product interacts both with Chs4p and Cdc10p and with one of the septins, Cdc10p; this analysis also revealed an interaction between Chs4p and Chs3p, the catalytic subunit of chitin synthase III. Bni4p has no known homologues; it contains a predicted coiled-coil domain, but no other recognizable motifs. Deletion of BNI4 is not lethal, but causes delocalization of chitin deposition and aberrant cellular morphology. Overexpression of Bni4p also causes delocalization of chitin deposition and produces a cellular morphology similar to that of septin mutants. Immunolocalization experiments show that Bni4p localizes to a ring at the mother-bud neck that lies predominantly on the mother-cell side (corresponding to the predominant site of chitin deposition). This localization depends on the septins but not on Chs4p or Chs3p. A GFP-Chs4p fusion protein also localizes to a ring at the mother-bud neck on the mother-cell side. This localization is dependent on the septins, Bni4p, and Chs3p. Chs3p, whose normal localization is similar to that of Chs4p, does not localize properly in bni4, chs4, or septin mutant strains or in strains that accumulate excess Bni4p. In contrast, localization of the septins is essentially normal in bni4, chs4, and chs3 mutant strains and in strains that accumulate excess Bni4p. Taken together, these results suggest that the normal localization of chitin synthase III activity is achieved by assembly of a complex in which Chs3p is linked to the septins via Chs4p and Bni4p.

Ultrastructural changes associated with the haustorial mother cell septum during haustorium formation inUromyces phaseoli var.vignae

The ultrastructural features of developing haustoria and associated haustorial mother cell septa were examined in the cowpea rust fungus,Uromyces phaseoli var.vignae. Significant changes occurred in the septal pore apparatus, and in structures associated with the septum, during the formation of a haustorium. The originally open septal pore became plugged with osmiophilic material and, on both sides of the septum, elaborations of the plasmalemma developed whose maximum growth coincided with the close association of numerous mitochondria. Those elaborations on the haustorial mother cell side consisted of irregular whorls and tangles of membranes and disappeared prior to the formation of the penetration peg. In contrast, the more tubular protrusions on the hyphal side of the septum reached their maximum length of about 900 nm during the breaching of the host cell wall but subsequently shrank rapidly until, by the time the haustorium was mature, they and their osmiophilic matrix adjacent to the septal wall had usually disappeared. By this time, the osmiophilic plug had also disappeared and the pore now resembled that most commonly seen in other portions of the vegetative mycelium in being covered on either side by a thin layer of osmiophilic material and in being surrounded by a region of differentiated cytoplasm bordered with microbodies. Little change in this septal pore apparatus was observed as the haustorium aged until the tonoplast of the haustorial mother cell broke down. At this time the differentiated regions of the cytoplasm, and the associated microbodies, disappeared from the haustorial mother cell side of the septum.

Budding and Metamorphosis in Acineta tuberosa. An Electron Microscopic Study on Morphogenesis in Suctoria

SYNOPSIS. All stages (except conjugation) of the life cycle in Acineta tuberosa (Suctoria) were studied in the electron microscope. Budding starts with the invagination of a small pellicular area near the opening of the vacuole and bearing rows of barren basal bodies. Some of these basal bodies are incorporated into the swarmer‐anlage and give rise to 8 rows of cilia, while the other basal bodies remain barren on the mother cell's side of the brood pouch. They will provide their own genetic continuity and later give rise to the swarmer of the next generation. Kineties are not spaced on the growing swarmer‐anlage until their cilia are fully grown. Further details on the stalk‐forming crganelle and general features of the development of the larvae are given.Metamorphosis starts with the formation of basal disc and stalk. The stalk‐forming material is released thru scopular pores into a tube‐like mold formed by the scopula after having been pulled inward. Later, scopula pores spread from the scopular area proper and move upward. On their way to the apical part of the animal the cuticle is secreted. The differentiation of tentacles is described and new observations regarding a possible mode of haptocyst formation are given.The results are discussed with respect to the morphogenetic potentialities of cortical structures in general.

真菌細胞の発育過程における微細構造の変化

Yeast-like cells (blastospores) of Candida albicans and C. stellatoidea present two types of cell wall formation in the process of cell growth. One type occurs in the bud formation, and the other in the germination. The present paper deals with the mechanism of these types of cell wall formation at the electron microscopical level.Bud formation occurred mostly when the yeast-like cells were cultured on Sabouraud's agar medium. The bud wall was produced by extension of the whole layers of the mother cell wall. After the bud cell developed fully in size, as well as in contents, the septal wall was formed in it, first on the mother-cell side and then on the bud-cell side. It was characteristic of electron-dense granules to be seen associated with membranous structures around the newly formed septal wall. These results suggested some possible mechanism for the septal-wall formation.The septal wall may be produced by the accumulation of structural substances activated by the aid of these granules observed, mitochondria, and other membranous structures. It may not be produced by an inward extension of the cell wall, nor by simple fission.The germination of blastospores took place easily with a diversity of unknown environmental factors. After the outer wall ruptured in the germinating region, the inner most layer of the cell wall, which was thin and transparent in electron density, protruded to be thick, so that an intact wall might develop in the daughter cell.In an elongated germ-tube, septal walls were observed. They were similar in appearance to those of mycelial fungi.It is interesting to note that the same type of cells (blastospores) takes a different growth phase depending upon the kind of culture.