SpoIIB localizes to active sites of septal biogenesis and spatially regulates septal thinning during engulfment in bacillus subtilis

A key step in the Bacillus subtilis spore formation pathway is the engulfment of the forespore by the mother cell, a phagocytosis-like process normally accompanied by the loss of peptidoglycan within the sporulation septum. We have reinvestigated the role of SpoIIB in engulfment by using the fluorescent membrane stain FM 4-64 and deconvolution microscopy. We have found that spoIIB mutant sporangia display a transient engulfment defect in which the forespore pushes through the septum and bulges into the mother cell, similar to the situation in spoIID, spoIIM, and spoIIP mutants. However, unlike the sporangia of those three mutants, spoIIB mutant sporangia are able to complete engulfment; indeed, by time-lapse microscopy, sporangia with prominent bulges were found to complete engulfment. Electron micrographs showed that in spoIIB mutant sporangia the dissolution of septal peptidoglycan is delayed and spatially unregulated and that the engulfing membranes migrate around the remaining septal peptidoglycan. These results demonstrate that mother cell membranes will move around septal peptidoglycan that has not been completely degraded and suggest that SpoIIB facilitates the rapid and spatially regulated dissolution of septal peptidoglycan. In keeping with this proposal, a SpoIIB-myc fusion protein localized to the sporulation septum during its biogenesis, discriminating between the site of active septal biogenesis and the unused potential division site within the same cell.     

Engulfment during sporulation in Bacillus subtilis is governed by a multi-protein complex containing tandemly acting autolysins

The conversion of a growing cell into an endospore in Bacillus subtilis involves a phagocytic-like process in which the developing spore (the forespore) is wholly engulfed by the adjacent mother cell. A prerequisite for engulfment is the removal of peptidoglycan from the septum that separates the forespore from the mother cell, a process that depends on the autolysin SpoIID and two proteins of unknown function, SpoIIM and SpoIIP. Here we present evidence that SpoIIP is also an autolysin, that it acts in tandem with SpoIID, and that all three proteins are in a complex with each other. We further show that the members of the complex exhibit a hierarchical relationship in which SpoIIM is responsible for localization to the septal membrane, SpoIIP localizes to the septal membrane by interacting with SpoIIM, and SpoIID, in turn, localizes by interacting with SpoIIP. Finally, we show that localization of SpoIIM depends on a fourth protein SpoIIB, raising the possibility that the complex contains an additional component and creating an overall hierarchy of the form: SpoIIB-->SpoIIM-->SpoIIP-->SpoIID.     

Forespore engulfment mediated by a ratchet-like mechanism

A key step in bacterial endospore formation is engulfment, during which one bacterial cell engulfs another in a phagocytosis-like process that normally requires SpoIID, SpoIIM, and SpoIIP (DMP). We here describe a second mechanism involving the zipper-like interaction between the forespore protein SpoIIQ and its mother cell ligand SpoIIIAH, which are essential for engulfment when DMP activity is reduced or SpoIIB is absent. They are also required for the rapid engulfment observed during the enzymatic removal of peptidoglycan, a process that does not require DMP. These results suggest the existence of two separate engulfment machineries that compensate for one another in intact cells, thereby rendering engulfment robust. Photobleaching analysis demonstrates that SpoIIQ assembles a stationary structure, suggesting that SpoIIQ and SpoIIIAH function as a ratchet that renders forward membrane movement irreversible. We suggest that ratchet-mediated engulfment minimizes the utilization of chemical energy during this dramatic cellular reorganization, which occurs during starvation.     

The SpoIIQ landmark protein has different requirements for septal localization and immobilization

Bacillus subtilis sporulation depends on the forespore membrane protein SpoIIQ, which interacts with the mother cell protein SpoIIIAH at the septum to localize other sporulation proteins. It has remained unclear how SpoIIQ localizes. We demonstrate that localization of SpoIIQ is achieved by two pathways: SpoIIIAH and the SpoIID, SpoIIM, SpoIIP engulfment proteins. SpoIIQ shows diffuse localization only in a mutant lacking both pathways. Super‐resolution imaging shows that in the absence of SpoIIIAH, SpoIIQ forms fewer, slightly larger foci than in wild type. Surprisingly, photobleaching experiments demonstrate that, although SpoIIQ localizes without SpoIIIAH, it is no longer immobilized, and is therefore able to exchange subunits within a localized pool. SpoIIQ mobility is further increased by the additional absence of the engulfment proteins. However an enzymatically inactive SpoIID protein immobilizes SpoIIQ even in the absence of SpoIIIAH, indicating that complete septal thinning is not required for SpoIIQ localization. This suggests that SpoIIQ interacts with both SpoIIIAH and the engulfment proteins or their peptidoglycan cleavage products. They further demonstrate that apparently normal localization of a protein without a binding partner can mask dramatic alterations in protein mobility. We speculate that SpoIIQ assembles foci along the path defined by engulfment proteins degrading peptidoglycan.     

Septal localization of forespore membrane proteins during engulfment in Bacillus subtilis

In Bacillus subtilis, many membrane proteins localize to the sporulation septum, where they play key roles in spore morphogenesis and cell-specific gene expression, but the mechanism for septal targeting is not well understood. SpoIIQ, a forespore-expressed protein, is involved in engulfment and forespore-specific gene expression. We find that SpoIIQ dynamically localizes to the sporulation septum, tracks the engulfing mother cell membrane, assembles into helical arcs around the forespore and is finally degraded. Retention of SpoIIQ in the septum requires one or more mother cell-expressed proteins. We also observed that any forespore-expressed membrane protein initially localizes to the septum and later spreads throughout the forespore membrane, suggesting that membrane protein insertion occurs at the forespore septal region. This possibility provides an attractive mechanism for how activation of mother cell-specific gene expression is restricted to adjacent sister cells, since direct insertion of the signaling protein SpoIIR into the septum would spatially restrict its activity. In keeping with this hypothesis, we find that SpoIIR localizes to the septum and is transiently expressed.     

Evidence that the SpoIIIE DNA translocase participates in membrane fusion during cytokinesis and engulfment

During Bacillus subtilis sporulation, SpoIIIE is required for translocation of the trapped forespore chromosome across the sporulation septum, for compartmentalization of cell-specific gene expression, and for membrane fusion after engulfment. We isolated mutations within the SpoIIIE membrane domain that block localization and function. One mutant protein initially localizes normally and completes DNA translocation, but shows reduced membrane fusion after engulfment. Fluorescence recovery after photobleaching experiments demonstrate that in this mutant the sporulation septum remains open, allowing cytoplasmic contents to diffuse between daughter cells, suggesting that it blocks membrane fusion after cytokinesis as well as after engulfment. We propose that SpoIIIE catalyses these topologically opposite fusion events by assembling or disassembling a proteinaceous fusion pore. Mutants defective in SpoIIIE assembly also demonstrate that the ability of SpoIIIE to provide a diffusion barrier is directly proportional to its ability to assemble a focus at the septal midpoint during DNA translocation. Thus, SpoIIIE mediates compartmentalization by two distinct mechanisms: the SpoIIIE focus first provides a temporary diffusion barrier during DNA translocation, and then mediates the completion of membrane fusion after division to provide a permanent diffusion barrier. SpoIIIE-like proteins might therefore serve to couple the final step in cytokinesis, septal membrane fusion, to the completion of chromosome segregation.     

Localization of RHO-4 indicates differential regulation of conidial versus vegetative septation in the filamentous fungus Neurospora crassa

Rho-4 mutants of the filamentous fungus Neurospora crassa lack septa and asexual spores (conidia) and grow slowly. In this report, localization of green fluorescent protein-tagged RHO-4 is used to elucidate the differences in factors controlling RHO-4 localization during vegetative growth versus asexual development. RHO-4 forms a ring at incipient vegetative septation sites that constricts with the formation of the septum toward the septal pore; RHO-4 persists around the septal pore after septum completion. During the formation of conidia, RHO-4 localizes to the primary septum but subsequently is relocalized to the cytoplasm after the placement of the secondary septum. Cytoplasmic localization and inactivation of RHO-4 are mediated by a direct physical interaction with RDI-1, a RHO guanosine nucleotide dissociation inhibitor. Inappropriate activation of the cyclic AMP-dependent protein kinase A pathway during vegetative growth causes mislocalization of RHO-4 away from septa to the cytoplasm, a process which was dependent upon RDI-1. An adenylate cyclase cr-1 mutant partially suppresses the aconidial defect of rho-4 mutants but only rarely suppresses the vegetative septation defect, indicating that conidial septation is negatively regulated by CR-1. These data highlight the differences in the regulation of septation during conidiation versus vegetative septation in filamentous fungi.     

Requirement for the cell division protein DivIB in polar cell division and engulfment during sporulation in Bacillus subtilis

During spore formation in Bacillus subtilis, cell division occurs at the cell pole and is believed to require essentially the same division machinery as vegetative division. Intriguingly, although the cell division protein DivIB is not required for vegetative division at low temperatures, it is essential for efficient sporulation under these conditions. We show here that at low temperatures in the absence of DivIB, formation of the polar septum during sporulation is delayed and less efficient. Furthermore, the polar septa that are complete are abnormally thick, containing more peptidoglycan than a normal polar septum. These results show that DivIB is specifically required for the efficient and correct formation of a polar septum. This suggests that DivIB is required for the modification of sporulation septal peptidoglycan, raising the possibility that DivIB either regulates hydrolysis of polar septal peptidoglycan or is a hydrolase itself. We also show that, despite the significant number of completed polar septa that form in this mutant, it is unable to undergo engulfment. Instead, hydrolysis of the peptidoglycan within the polar septum, which occurs during the early stages of engulfment, is incomplete, producing a similar phenotype to that of mutants defective in the production of sporulation-specific septal peptidoglycan hydrolases. We propose a role for DivIB in sporulation-specific peptidoglycan remodelling or its regulation during polar septation and engulfment.     

Synaptic targeting of PSD-Zip45 (Homer 1c) and its involvement in the synaptic accumulation of F-actin

PSD-Zip45/Homer1c, which contains an enabled/VASP homology 1 (EVH1) domain and leucine zipper motifs, is a postsynaptic density (PSD) scaffold protein that interacts with metabotropic glutamate receptors and the shank family. We studied the molecular mechanism underlying the synaptic targeting of PSD-Zip45 in cultured hippocampal neurons. The EVH1 domain and the extreme C-terminal leucine zipper motif were molecular determinants for its synaptic targeting. The overexpression of the mutant of the EVH1 domain or deletion of the extreme C-terminal leucine zipper motif markedly suppressed the synaptic localization of endogenous shank but not PSD-95 or GKAP. In contrast, an overexpressed GKAP mutant lacking shank binding activity had no effect on the synaptic localization of shank. Actin depolymerization by latrunculin A reduced the synaptic localization of PSD-Zip45, shank, and F-actin but not of PSD-95 or GKAP. Overexpression of PSD-Zip45 enhanced the accumulation of synaptic F-actin. Additionally, overexpression of PSD-Zip45 and an isoform of shank induced synaptic enlargement in association with the further accumulation of synaptic F-actin. The EVH1 domain and extreme C-terminal leucine zipper motif of PSD-Zip45 were also critical for these events. Thus, these data suggest that the PSD-Zip45-shank and PSD-95-GKAP complexes form different synaptic compartments, and PSD-Zip45 alone or PSD-Zip45-shank is involved in the synaptic accumulation of F-actin.     

The pathogenesis of atrial and atrioventricular septal defects with special emphasis on the role of the dorsal mesenchymal protrusion

Partitioning of the four-chambered heart requires the proper formation, interaction and fusion of several mesenchymal tissues derived from different precursor populations that together form the atrioventricular mesenchymal complex. This includes the major endocardial cushions and the mesenchymal cap of the septum primum, which are of endocardial origin, and the dorsal mesenchymal protrusion (DMP), which is derived from the Second Heart Field. Failure of these structures to develop and/or fully mature results in atrial septal defects (ASDs) and atrioventricular septal defects (AVSD). AVSDs are congenital malformations in which the atria are permitted to communicate due to defective septation between the inferior margin of the septum primum and the atrial surface of the common atrioventricular valve. The clinical presentation of AVSDs is variable and depends on both the size and/or type of defect; less severe defects may be asymptomatic while the most severe defect, if untreated, results in infantile heart failure. For many years, maldevelopment of the endocardial cushions was thought to be the sole etiology of AVSDs. More recent work, however, has demonstrated that perturbation of DMP development also results in AVSD. Here, we discuss in detail the formation of the DMP, its contribution to cardiac septation and describe the morphological features as well as potential etiologies of ASDs and AVSDs.     

SpoIID-Mediated Peptidoglycan Degradation Is Required throughout Engulfment during Bacillus subtilis Sporulation

SpoIID is a membrane-anchored enzyme that degrades peptidoglycan and is essential for engulfment and sporulation in Bacillus subtilis. SpoIID is targeted to the sporulation septum, where it interacts with two other proteins required for engulfment: SpoIIP and SpoIIM. We changed conserved amino acids in SpoIID to alanine to determine whether there was a correlation between the effect of each substitution on the in vivo and in vitro activities of SpoIID. We identified one amino acid substitution, E88A, that eliminated peptidoglycan degradation activity and one, D210A, that reduced it, as well as two substitutions that destabilized the protein in B. subtilis (R106A and K203A). Using these mutants, we show that the peptidoglycan degradation activity of SpoIID is required for the first step of engulfment (septal thinning), as well as throughout membrane migration, and we show that SpoIID levels are substantially above the minimum required for engulfment. The inactive mutant E88A shows increased septal localization compared to the wild type, suggesting that the degradation cycle of the SpoIID/SpoIIP complex is accompanied by the activity-dependent release of SpoIID from the complex and subsequent rebinding. This mutant is also capable of moving SpoIIP across the sporulation septum, suggesting that SpoIID binding, but not peptidoglycan degradation activity, is needed for relocalization of SpoIIP. Finally, the mutant with reduced activity (D210A) causes uneven engulfment and time-lapse microscopy indicates that the fastest-moving membrane arm has greater concentrations of SpoIIP than the slower-moving arm, demonstrating a correlation between SpoIIP protein levels and the rate of membrane migration.Endospore formation is an evolutionarily conserved process that allows Bacillus subtilis and related Gram-positive bacteria to adapt to changes in the environment, such as nutrient depletion. Many dramatic morphological changes occur during sporulation, each requiring a multitude of specialized proteins (reviewed in references 13 and 17). First, a sporulation septum is formed near one of the cell poles, forming two separate compartments of unequal sizes and with differing fates (Fig. ​(Fig.1A).1A). The smaller of the two, the forespore, will eventually become the spore, while the larger, the mother cell, will ultimately lyse. Next, the mother cell membranes move up and around the forespore in the poorly understood process of engulfment. Although this process is superficially similar to eukaryotic engulfment, it is complicated by the thick cell wall that surrounds and separates the two compartments. After engulfment, the migrating membranes pinch off from the mother cell membrane, thereby releasing the forespore into the cytoplasm of the mother cell, where it can be enveloped with protective coat proteins and eventually released into the environment as a mature spore. Sporulation provides an ideal, nonessential system for understanding how bacterial cells are capable of undergoing dramatic morphological changes.Open in a separate windowFIG. 1.Engulfment in B. subtilis. (A) (i) Engulfment begins with formation of an asymmetric septum that divides the cell into the forespore (FS) and mother cell (MC). SpoIID (orange pacman) and SpoIIP (green pacman) peptidoglycan degradation enzymes localize to the center of the septum. (ii) SpoIID and SpoIIP thin the septal peptidoglycan, starting from the center and moving toward the cell edges. SpoIIQ (purple ball) and SpoIIIAH (red ball) form a zipper across the septum, assembling foci behind the leading edges. (iii) The peptidoglycan degradation enzymes localize to the leading edges during membrane migration, while additional SpoIIQ-SpoIIIAH complexes assemble around the forespore. (iv) Engulfment membrane fission occurs at the top of the forespore, releasing the forespore into the mother cell cytoplasm. (B) Burnt-bridge Brownian ratchet model for membrane migration, adapted from earlier studies (1, 7). (C) Schematic representation of the SpoIID domain structure. The transmembrane domain (TM) and putative enzymatic domain, as defined by Pfam (14), are indicated. Amino acid numbers are below the schematic, and mutations causing in vivo phenotypes are indicated by an “X”.Engulfment involves dynamic protein localization and large-scale rearrangements of cellular membranes and peptidoglycan to accommodate internalization of the forespore. The physical basis for engulfment remains unclear, but two separate protein machineries that contribute to engulfment have been discovered. The first module involves the only three proteins known to be required for engulfment under all physiological conditions: SpoIID, SpoIIM, and SpoIIP (16, 24, 35). Zymography assays have demonstrated that both SpoIID and SpoIIP degrade peptidoglycan in vitro (1, 8), and this function is thought to be essential for engulfment in wild-type cells (1, 2, 8). SpoIID and SpoIIP are membrane-spanning proteins that directly interact both in vivo and in vitro, as demonstrated by coimmunoprecipitation and affinity chromatography techniques (2, 8). These studies failed to demonstrate an interaction between SpoIIM and either SpoIID or SpoIIP, perhaps because SpoIIM is an integral membrane protein. However, SpoIIM is required for localization of SpoIID and SpoIIP (2, 8), suggesting that all three proteins interact to form a peptidoglycan degradation module that is essential for engulfment.The second system influencing membrane migration is the SpoIIQ/SpoIIIAH zipper, which is required for engulfment only under certain conditions (2, 7, 38). SpoIIQ is produced in the forespore (23) and SpoIIIAH is produced in the mother cell (19). SpoIIQ and SpoIIIAH interact both in vitro and in vivo via their extracellular domains (6, 7, 10). Because these two proteins are produced in separate compartments, the only possible place for an interaction is the intermembrane space between the mother cell and forespore, forming a protein-protein zipper between the two cells. This zipperlike interaction is necessary for septal localization of SpoIIIAH and other mother cell proteins (6, 10, 18) and is capable of holding the two cells together when peptidoglycan is removed with lysozyme (7). Surprisingly, digestion of the peptidoglycan with lysozyme also allows membrane migration in about half of treated cells, in a process requiring the SpoIIQ/SpoIIIAH zipper but not the SpoIIDMP peptidoglycan degradation module. The SpoIIQ-SpoIIIAH zipper also contributes to engulfment in living cells, since strains lacking SpoIIQ or SpoIIIAH complete engulfment more slowly than the wild type and have synergistic engulfment defects when certain secondary mutations are introduced (2, 7, 38). Together, these results strongly support a role for the SpoIIQ/SpoIIIAH module in engulfment, demonstrating that the zipper contributes to the efficiency of membrane migration even when the SpoIIDMP module is present and functional. They also suggest that the engulfment machinery displays functional redundancy and that the zipper module provides a backup machinery for membrane migration.The precise role of the SpoIIDMP module during engulfment remains unclear. One model proposes that SpoIID and SpoIIP act as a burnt-bridge Brownian ratchet (Fig. ​(Fig.1B)1B) (1, 7). This model asserts that as SpoIID and SpoIIP degrade peptidoglycan, they eliminate their own enzymatic targets, resulting in the absence of substrate in one direction and therefore, overall movement in the opposite direction. As the enzymes move forward toward new targets, the mother cell membranes are dragged along with them because they are anchored in the membrane. This hypothesis predicts that SpoIID and SpoIIP are processive enzymes and that the SpoIIDMP complex could function as a motor, moving along peptidoglycan as a track and pulling the membranes with it (1, 7). A second model predicts that peptidoglycan degradation could simply remove a steric hindrance to membrane migration (such as links between the forespore membrane and the cell wall) and that some other mechanism provides the force required for membrane migration. Although the SpoIIQ-SpoIIIAH module can contribute to membrane migration, these proteins are not always essential for engulfment in intact cells (7, 38), suggesting that another unidentified system must generate the force required for membrane movement if the DMP module does not act as a burnt-bridge Brownian ratchet. Recent evidence suggests that peptidoglycan biosynthesis, which is localized to the leading edge of the engulfing membrane and necessary for membrane migration in the absence of the SpoIIQ-SpoIIIAH proteins, might be this missing force generating mechanism (26).Both models predict that the activities of SpoIID and SpoIIP are essential for membrane migration. This requirement has been demonstrated for SpoIIP (8) and, while this work was under review, for SpoIID. SpoIID shows no sequence similarity to any characterized enzyme that degrades peptidoglycan and thus constitutes the founding member of a new class of enzymes that remodel peptidoglycan (1, 27). However, SpoIID does show some similarity to B. subtilis LytB (24), a protein that enhances the activity of the amidase LytC (5, 20, 34), while SpoIIP is related to LytC (14). A recent study demonstrated that SpoIIP is both an amidase and endopeptidase and that SpoIID both activates SpoIIP and functions as a lytic transglycosylase, cleaving peptidoglycan between NAG and NAM (27). Together, these two enzymes degrade peptidoglycan into its smallest repeating subunits. However, it remains unclear which of the demonstrated or suggested biochemical functions of SpoIID are required for its various in vivo activities (interaction with SpoIIP, localization, septal thinning, and membrane migration), and it is unclear whether peptidoglycan degradation activity is required throughout engulfment or only for the initial stage of septal thinning.We use site-directed mutagenesis to test the role of 56 conserved amino acids in SpoIID, focusing on hydrophilic amino acids that might be involved in protein-protein interactions and peptidoglycan degradation. We identified one mutation (E88A) that eliminates and three others (R106A, K203A, and D210A) that reduce peptidoglycan degradation activity and show that SpoIID activity is required for the earliest stage of engulfment (septal thinning), as well as throughout membrane migration. Our results confirm and extend those of Morlot et al. (27) and also demonstrate that SpoIID activity is required throughout engulfment. Furthermore, our data indicate that the enzymatically inactive mutant protein (E88A) shows increased septal localization compared to the wild-type protein, suggesting that peptidoglycan degradation contributes to the release of SpoIID from the septum. We propose a modified model for the enzymatic cycle of the SpoIID and SpoIIP complex.     

p62 Targeting to the autophagosome formation site requires self-oligomerization but not LC3 binding

Autophagy is an intracellular degradation process by which cytoplasmic contents are degraded in the lysosome. In addition to nonselective engulfment of cytoplasmic materials, the autophagosomal membrane can selectively recognize specific proteins and organelles. It is generally believed that the major selective substrate (or cargo receptor) p62 is recruited to the autophagosomal membrane through interaction with LC3. In this study, we analyzed loading of p62 and its related protein NBR1 and found that they localize to the endoplasmic reticulum (ER)-associated autophagosome formation site independently of LC3 localization to membranes. p62 colocalizes with upstream autophagy factors such as ULK1 and VMP1 even when autophagosome formation is blocked by wortmannin or FIP200 knockout. Self-oligomerization of p62 is essential for its localization to the autophagosome formation site. These results suggest that p62 localizes to the autophagosome formation site on the ER, where autophagosomes are nucleated. This process is similar to the yeast cytoplasm to vacuole targeting pathway.     

A Fail-Safe Mechanism in the Septal Ring Assembly Pathway Generated by the Sequential Recruitment of Cell Separation Amidases and Their Activators

During cytokinesis in Escherichia coli, the peptidoglycan (PG) layer produced by the divisome must be split to promote cell separation. Septal PG splitting is mediated by the amidases: AmiA, AmiB, and AmiC. To efficiently hydrolyze PG, the amidases must be activated by LytM domain factors. EnvC specifically activates AmiA and AmiB, while NlpD specifically activates AmiC. Here, we used an exportable, superfolding variant of green fluorescent protein (GFP) to demonstrate that AmiB, like its paralog AmiC, is recruited to the division site by an N-terminal targeting domain. The results of colocalization experiments indicate that EnvC is recruited to the division site well before its cognate amidase AmiB. Moreover, we show that EnvC and AmiB have differential FtsN requirements for their localization. EnvC accumulates at division sites independently of this essential division protein, whereas AmiB localization is FtsN dependent. Interestingly, we also report that AmiB and EnvC are recruited to division sites independently of one another. The same is also true for AmiC and NlpD. However, unlike EnvC, we find that NlpD shares an FtsN-dependent localization with its cognate amidase. Importantly, when septal PG synthesis is blocked by cephalexin, both EnvC and NlpD are recruited to septal rings, whereas the amidases fail to localize. Our results thus suggest that the order in which cell separation amidases and their activators localize to the septal ring relative to other components serves as a fail-safe mechanism to ensure that septal PG synthesis precedes the expected burst of PG hydrolysis at the division site, accompanied by amidase recruitment.     

Phosphatidylethanolamine domains and localization of phospholipid synthases in Bacillus subtilis membranes

Application of the cardiolipin (CL)-specific fluorescent dye 10-N-nonyl-acridine orange has recently revealed CL-rich domains in the septal regions and at the poles of the Bacillus subtilis membrane (F. Kawai, M. Shoda, R. Harashima, Y. Sadaie, H. Hara, and K. Matsumoto, J. Bacteriol. 186:1475-1483, 2004). This finding prompted us to examine the localization of another phospholipid, phosphatidylethanolamine (PE), with the cyclic peptide probe, Ro09-0198 (Ro), that binds specifically to PE. Treatment with biotinylated Ro followed by tetramethyl rhodamine-conjugated streptavidin revealed that PE is localized in the septal membranes of vegetative cells and in the membranes of the polar septum and the engulfment membranes of sporulating cells. When the mutant cells of the strains SDB01 (psd1::neo) and SDB02 (pssA10::spc), which both lack PE, were examined under the same conditions, no fluorescence was observed. The localization of the fluorescence thus evidently reflected the localization of PE-rich domains in the septal membranes. Similar PE-rich domains were observed in the septal regions of the cells of many Bacillus species. In Escherichia coli cells, however, no PE-rich domains were found. Green fluorescent protein fusions to the enzymes that catalyze the committed steps in PE synthesis, phosphatidylserine synthase, and in CL synthesis, CL synthase and phosphatidylglycerophosphate synthase, were localized mainly in the septal membranes in B. subtilis cells. The majority of the lipid synthases were also localized in the septal membranes; this includes 1-acyl-glycerol-3-phosphate acyltransferase, CDP-diacylglycerol synthase, phosphatidylserine decarboxylase, diacylglycerol kinase, glucolipid synthase, and lysylphosphatidylglycerol synthase. These results suggest that phospholipids are produced mostly in the septal membranes and that CL and PE are kept from diffusing out to lateral ones.     

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.     

Mechanisms of internalization of apoptotic and necrotic L929 cells by a macrophage cell line studied by electron microscopy

Rapid and efficient phagocytic removal of dying cells is a key feature of apoptosis. In necrotic caspase-independent modes of death, the role and extent of phagocytosis is not well documented. To address this issue, we studied at the ultrastructural level the phagocytic response to dying cells in an in vitro phagocytosis assay with a mouse macrophage cell line (Mf4/4). As target cells, murine L929sAhFas cells were induced to die by TNFR1-mediated necrosis or by Fas-mediated apoptosis. Apoptotic L929sAhFas cells are taken up by complete engulfment of apoptotic bodies as single entities forming a tight-fitting phagosome, thus resembling the "zipper"-like mechanism of internalization. In contrast, primary and secondary necrotic cells were internalized by a macropinocytotic mechanism with formation of multiple ruffles by the ingesting macrophage. Ingestion of necrotic cellular material was invariably taking place after the integrity of the cell membrane was lost and did not occur as discrete particles, in contrast to apoptotic material that is surrounded by an intact membrane. Although nuclei of necrotic cells have been observed in the vicinity of macrophages, no uptake of necrotic nuclei was observed. The present report provides a basis for future studies aimed at discovering molecular pathways that precede these diverse mechanisms of uptake.