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We present two distinct, complementary methods for improving axial resolution in three-dimensional structured illumination microscopy (3D SIM) with minimal or no modification to the optical system. First, we show that placing a mirror directly opposite the sample enables 4-beam interference with higher spatial frequency content than 3D SIM illumination, offering near-isotropic imaging with ∼120 nm lateral and 160 nm axial resolution. Second, we develop an improved deep learning method that can be directly applied to 3D SIM data, obviating the need for additional hardware. This procedure results in ∼120 nm isotropic resolution and can be combined with denoising to facilitate volumetric imaging spanning dozens of time points. We demonstrate the potential of these advances by imaging a variety of cellular samples, delineating the nanoscale distribution of vimentin and microtubule filaments, observing the relative positions of caveolar coat proteins and lysosomal markers, and visualizing rich cytoskeletal dynamics within T-cells in the early stages of immune synapse formation.

Bacterial spores are dormant cells that are encased in a thick protein shell, the “coat,” which participates in protecting the organism’s DNA from environmental insults. The coat is composed of dozens of proteins that assemble in an orchestrated fashion during sporulation. In Bacillus subtilis, 2 proteins initiate coat assembly: SpoVM, which preferentially binds to micron-scale convex membranes and marks the surface of the developing spore as the site for coat assembly; and SpoIVA, a structural protein recruited by SpoVM that uses ATP hydrolysis to drive its irreversible polymerization around the developing spore. Here, we describe the initiation of coat assembly by SpoVM and SpoIVA. Using single-molecule fluorescence microscopy in vivo in sporulating cells and in vitro on synthetic spores, we report that SpoVM’s localization is primarily driven by a lower off-rate on membranes of preferred curvature in the absence of other coat proteins. Recruitment and polymerization of SpoIVA results in the entrapment of SpoVM on the forespore surface. Using experimentally derived reaction parameters, we show that a 2-dimensional ratchet model can describe the interdependent localization dynamics of SpoVM and SpoIVA, wherein SpoVM displays a longer residence time on the forespore surface, which favors recruitment of SpoIVA to that location. Localized SpoIVA polymerization in turn prevents further sampling of other membranes by prelocalized SpoVM molecules. Our model therefore describes the dynamics of structural proteins as they localize and assemble at the correct place and time within a cell to form a supramolecular complex.

We carry out Molecular Dynamics (MD) simulations to compare the equilibrium architecture and properties of nanoparticle-supported lipid bilayers (NPSLBLs) with the free vesicles of similar dimensions. Three key differences emerge. Firstly, we witness that for a free vesicle, a much larger number of lipid molecules occupy the outer layer as compared to the inner layer; on the other hand, for the NPSLBL the number of lipid molecules occupying the inner and outer layers is identical. Secondly, we witness that the diffusivities of the lipid molecules occupying both the inner and the outer layers of the free vesicles are identical, whereas for the NPSLBLs the diffusivity of the lipid molecules in the outer layer is more than twice the diffusivity of the lipid molecules in the inner layer. Finally, the NPSLBLs entrap nanoscopic thin water film between the inner lipid layer and the NP and the diffusivity of this water film is nearly one order of magnitude smaller than the diffusivity of the bulk water; on the other hand, the water inside the free vesicles has a diffusivity that is only slightly lower than that of the bulk water. Our findings, possibly the first probing the atomistic details of the NPSLBLs, are anticipated to shed light on the properties of this important nanomaterial with applications in a large number of disciplines ranging from drug and gene delivery to characterizing curvature-sensitive molecules.

The overexpression of the cell surface receptor “HER-2” is a common marker for many breast and ovarian cancers. New treatments for these cancers involve the use of monoclonal antibodies directed against HER2. SSHELs are synthetic bacterial spore-like particles wherein the spore’s cell surface is partially reconstituted around 1 µm-diameter silica beads. Via a unique cysteine engineered protein, the surface of SSHELs may be covalently decorated with molecules of interest. We showed that SSHELs decorated with anti-HER2 affibodies (SSHELs∝HER2) specifically bound to target cells and were internalized. Doxorubicin loaded SSHELs (Dox-SSHELs∝HER2) were internalized and trafficked to lysosomes, whereupon the cargo was released in a pH-dependent manner. Dox-SSHELs∝HER2 reduce tumor growth with lower toxicity in a mouse tumor xenograft model when compared to the free drug. Further, when compared to an FDA approved liposome-encased doxorubicin (AD), Dox-SSHELs∝HER2 showed similar efficacy in reducing tumor growth, but without any of the typical side effects correlated with AD injections (including weight loss, tumor ulceration, skin descaling). SKOV3 and SKBR3 cells’ targeting by SSHELs∝HER2 was evaluated by flow cytometry and confocal microscopy. Dox-SSHELs∝HER2 anti-tumor effect was evaluated in vitro by flow cytometry and caspase assay. HER2- cells were used as a control. Dox-SSHELs∝HER2’s efficacy was tested in vivo using athymic nude mice injected with SKOV3 cells and then treated for up to 40 days. SSHELs∝HER2 preferentially target cancer cells proportionally to their concentration, as flow cytometry showed. Additional analysis showed the inability of SSHELs∝HER2 to bind HER2+ cells if the receptor was previously blocked. Delivery-mechanism and co-localization analysis showed that SSHELs∝HER2 are preferentially taken up using macropinocytosis and that they undergo a lysosomal pathway where acidic pH contributes to release the cargo. Dox-SSHELs∝HER2 reduce the viability of the treated samples inducing DNA damages and triggering caspases with increase efficiency towards HER2+ cells. In vivo, we demonstrated that Dox-SSHELs∝HER2 can reduce SKOV3 tumor growth up to 75% when compared to the free drug. Additionally, the doxorubicin delivered by SSHELs∝HER2 did not lead to any side effect compared to the free drug or AD. Although new therapies are rising and becoming available for cancer treatments, limited options are still available. We suggest that SSHELs provide several unique benefits and that they may be used as an additional strategy for this purpose. With a facile, reproducible manufacturing process that allows for straightforward targeting towards specific cell types, pH driven cargo release, and lower overall toxicity compared to a leading treatment, we suggest that SSHELs may represent a versatile strategy opening new research avenues for targeted drug delivery treatment of breast and ovarian cancer. Citation Format: Domenico D'Atri, Minsuk Kong, Maria Teresa Bilotta, Kandice Tanner, David J. Fitzgerald, Kumaran S. Ramamurthi. Cell-specific cargo delivery using synthetic bacterial spores [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1739.

Hydrolysis of nucleoside triphosphates releases similar amounts of energy. However, ATP hydrolysis is typically used for energy-intensive reactions, whereas GTP hydrolysis typically functions as a switch. SpoIVA is a bacterial cytoskeletal protein that hydrolyzes ATP to polymerize irreversibly during Bacillus subtilis sporulation. SpoIVA evolved from a TRAFAC class of P-loop GTPases, but the evolutionary pressure that drove this change in nucleotide specificity is unclear. We therefore reengineered the nucleotide-binding pocket of SpoIVA to mimic its ancestral GTPase activity. SpoIVAGTPase functioned properly as a GTPase but failed to polymerize because it did not form an NDP-bound intermediate that we report is required for polymerization. Further, incubation of SpoIVAGTPase with limiting ATP did not promote efficient polymerization. This approach revealed that the nucleotide base, in addition to the energy released from hydrolysis, can be critical in specific biological functions. We also present data suggesting that increased levels of ATP relative to GTP at the end of sporulation was the evolutionary pressure that drove the change in nucleotide preference in SpoIVA., eLife digest Living organisms need energy to stay alive; in cells, this energy is supplied in the form of a small molecule called adenosine triphosphate, or ATP, a nucleotide that stores energy in the bonds between its three phosphate groups. ATP is present in all living cells and is often referred to as the energy currency of the cell, because it can be easily stored and transported to where it is needed. However, it is unknown why cells rely so heavily on ATP when a highly similar nucleotide called guanosine triphosphate, or GTP, could also act as an energy currency. There are several examples of proteins that originally used GTP and have since evolved to use ATP, but it is not clear why this switch occurred. One suggestion is that ATP is the more readily available nucleotide in the cell. To test this hypothesis, Updegrove, Harke et al. studied a protein that helps bacteria transition into spores, which are hardier and can survive in extreme environments until conditions become favorable for bacteria to grow again. In modern bacteria, this protein uses ATP to provide energy, but it evolved from an ancestral protein that used GTP instead. First, Updegrove, Harke et al. engineered the protein so that it became more similar to the ancestral protein and used GTP instead of ATP. When this was done, the protein gained the ability to break down GTP and release energy from it, but it no longer performed its enzymatic function. This suggests that both the energy released and the source of that energy are important for a protein’s activity. Further analysis showed that the modern version of the protein has evolved to briefly hold on to ATP after releasing its energy, which did not happen with GTP in the modified protein. Updegrove, Harke et al. also discovered that the levels of GTP in a bacterial cell fall as it transforms into a spore, while ATP levels remain relatively high. This suggests that ATP may indeed have become the source of energy of choice because it was more available. These findings provide insights into how ATP became the energy currency in cells, and suggest that how ATP is bound by proteins can impact a protein’s activity. Additionally, these experiments could help inform the development of drugs targeting proteins that bind nucleotides: it may be essential to consider the entirety of the binding event, and not just the release of energy.

We have carried out coarse-grained molecular dynamics (MD) simulations to study the self-assembly procedure of a system of randomly placed lipid molecules, water beads, and a nanoparticle (NP). The self-assembly results in the formation of the nanoparticle-supported lipid bilayer (NPSLBL), with the self-assembly mechanism being driven by events such as the formation of small lipid clusters, merging of the lipid clusters in the vicinity of the NP to form NP-embedded vesicle with a pore, and collapsing of that pore to eventually form the equilibrated NPSLBL system overcoming a large free energy barrier. Subsequently, we quantify the properties and the configurations of this NPSLBL system. We reveal that unlike our proposition of equal number of lipid molecules occupying the inner and outer leaflets in a recent report studying the properties of preassembled lipid bilayer, the equilibrated self-assembled NPSLBL system demonstrates a much larger number of lipid molecules occupying the outer leaflet as compared to the inner leaflet. Secondly, the thickness of the water layer entrapped between the NP and the inner leaflet shows similar values as that predicted by experiments and our previous study. Finally, we reveal that, similar to our previous study, the diffusivity of the lipid molecules in the outer leaflet is larger than that in the inner leaflet but, due to higher temperature employed during our simulations, are even larger than that predicted by our previous study.

SSHELs are synthetic bacterial spore-like particles wherein the spore’s cell surface is partially reconstituted around 1 µm-diameter silica beads coated with a lipid bilayer. Via a unique cysteine engineered in one of the surface proteins, the surface of SSHELs may be covalently decorated with molecules of interest. Here, we modified SSHELs with an affibody directed against HER2, a cell surface protein overexpressed in some breast and ovarian cancer cells, and loaded them with the chemotherapeutic agent doxorubicin. Drug-loaded SSHELs reduced tumor growth with lower toxicity in a mouse tumor xenograft model compared to free drug by specifically binding to HER2-positive cancer cells. We show that SSHELs bound to target cells are taken up and trafficked to acidic compartments, whereupon the cargo is released in a pH-dependent manner. Finally, we demonstrate that SHELLs can clear small tumor lesions in a complex tumor microenvironment in a zebrafish model of brain metastasis. We propose that SSHELs represent a versatile strategy for targeted drug delivery.

Cytoskeletal proteins are classified as a group that is defined functionally, whose members are capable of polymerizing into higher order structures, either dynamically or statically, to perform structural roles during a variety of cellular processes. In eukaryotes, the most well-studied cytoskeletal proteins are actin, tubulin, and intermediate filaments, and are essential for cell shape and movement, chromosome segregation, and intracellular cargo transport. Prokaryotes often harbor homologs of these proteins, but in bacterial cells, these homologs are usually not employed in roles that can be strictly defined as ‘cytoskeletal’. However, several bacteria encode other proteins capable of polymerizing which, although they do not appear to have a eukaryotic counterpart, nonetheless appear to perform a more traditional ‘cytoskeletal’ function. In this review, we discuss recent reports that cover the structures and functions of prokaryotic proteins that are broadly termed as cytoskeletal, either by sequence homology or by function, to highlight how the enzymatic properties of traditionally studied cytoskeletal proteins may be used for other types of cellular functions; and to demonstrate how truly ‘cytoskeletal’ functions may be performed by uniquely bacterial proteins that do not display homology to eukaryotic proteins.

The use of quality control mechanisms to stall developmental pathways or completely remove defective cells from a population is a widespread strategy to ensure the integrity of morphogenetic programs. Endospore formation (sporulation) is a well conserved microbial developmental strategy in the Firmicutes phylum wherein a progenitor cell that faces starvation differentiates to form a dormant spore. Despite the conservation of this strategy, it has been unclear what selective pressure maintains the fitness of this developmental program, composed of hundreds of unique genes, during multiple rounds of vegetative growth when sporulation is not required. Recently, a quality control pathway was discovered in Bacillus subtilis which monitors the assembly of the spore envelope and specifically eliminates, through cell lysis, sporulating cells that assemble the envelope incorrectly. Here, we review the use of checkpoints that govern the entry into sporulation in B. subtilis and discuss how the use of regulated cell death pathways during bacterial development may help maintain the fidelity of the sporulation program in the species.

Flip-flop of lipids of the lipid bilayer (LBL) constituting the plasma membrane (PM) plays a crucial role in a myriad of events ranging from cellular signaling and regulation of cell shapes to cell homeostasis, membrane asymmetry, phagocytosis, and cell apoptosis. While extensive research has been conducted to probe the lipid flip flop of planar lipid bilayers (LBLs), less is known regarding lipid flip-flop for highly curved, nanoscopic LBL systems despite the vast importance of membrane curvature in defining the morphology of cells and organelles and in maintaining a variety of cellular functions, enabling trafficking, and recruiting and localizing shape-responsive proteins. In this paper, we conduct molecular dynamics (MD) simulations to study the energetics, structure, and configuration of a lipid molecule undergoing flip-flop and desorption in a highly curved LBL, represented as a nanoparticle-supported lipid bilayer (NPSLBL) system. We compare our findings against those of a planar substrate supported lipid bilayer (PSSLBL). Our MD simulation results reveal that despite the vast differences in the curvature and other curvature-dictated properties (e.g., lipid packing fraction, difference in the number of lipids between inner and outer leaflets, etc.) between the NPSLBL and the PSSLBL, the energetics of lipid flip-flop and lipid desorption as well as the configuration of the lipid molecule undergoing lipid flip-flop are very similar for the NPSLBL and the PSSLBL. In other words, our results establish that the curvature of the LBL plays an insignificant role in lipid flip-flop and desorption.

Binary fission has been well studied in rod-shaped bacteria, but the mechanisms underlying cell division in spherical bacteria are poorly understood. Rod-shaped bacteria harbor regulatory proteins that place and remodel the division machinery during cytokinesis. In the spherical human pathogen Staphylococcus aureus, we found that the essential protein GpsB localizes to mid-cell during cell division and co-constricts with the division machinery. Depletion of GpsB arrested cell division and led to cell lysis, whereas overproduction of GpsB inhibited cell division and led to the formation of enlarged cells. We report that S. aureus GpsB, unlike other Firmicutes GpsB orthologs, directly interacts with the core divisome component FtsZ. GpsB bundles and organizes FtsZ filaments and also stimulates the GTPase activity of FtsZ. We propose that GpsB orchestrates the initial stabilization of the Z-ring at the onset of cell division and participates in the subsequent remodeling of the divisome during cytokinesis.

Spores of Bacillus subtilis are encased in a protein coat composed of ∼80 different proteins. Recently, we reconstituted the basement layer of the coat, composed of two structural proteins (SpoVM and SpoIVA) around spore-sized silica beads encased in a lipid bilayer, to create synthetic spore-like particles termed ‘SSHELs’. We demonstrated that SSHELs could display thousands of copies of proteins and small molecules of interest covalently linked to SpoIVA. In this study, we investigated the efficacy of SSHELs in delivering vaccines. We show that intramuscular vaccination of mice with undecorated one micron-diameter SSHELs elicited an antibody response against SpoIVA. We further demonstrate that SSHELs covalently modified with a catalytically inactivated staphylococcal alpha toxin variant (Hla(H35L)), without an adjuvant, resulted in improved protection against Staphylococcus aureus infection in a bacteremia model as compared to vaccination with the antigen alone. Although vaccination with either Hla(H35L) or Hla(H35L) conjugated to SSHELs similarly elicited the production of neutralizing antibodies to Hla, we found that a subset of memory T cells was differentially activated when the antigen was delivered on SSHELs. We propose that the particulate nature of SSHELs elicits a more robust immune response to the vaccine that results in superior protection against subsequent S. aureus infection.

Binary fission has been well studied in rod-shaped bacteria, but the mechanisms underlying cell division in spherical bacteria are poorly understood. Rod-shaped bacteria harbor regulatory proteins that place and remodel the division machinery during cytokinesis. In the spherical human pathogen

Many essential cellular processes including endocytosis and vesicle trafficking require alteration of membrane geometry. These changes are usually mediated by proteins that can sense and/or induce membrane curvature. Using spherical nanoparticle supported lipid bilayers (SSLBs), we characterize how SpoVM, a bacterial development factor, interacts with differently curved membranes by magic angle spinning solid-state NMR. Our results demonstrate that SSLBs are an effective system for structural and topological studies of membrane geometry-sensitive molecules.

The last three decades have witnessed an explosion in mechanistic details on how model bacterial organisms such as Escherichia coli, Bacillus subtilis, and Caulobacter crescentus undergo binary fission. These advances were possible by not only advances in microscopy that allowed cell biological questions to be answered, but also by the clever use of genetic manipulations in these systems in which specific hypothesis could be directly and easily tested. More recently, research using traditionally understudied organisms, or “non-model” systems, has revealed several alternate mechanistic strategies that bacteria use to divide and propagate. In this review, we will highlight these new findings and compare these strategies to cell division mechanisms elucidated in well-established model organisms.

Summary In Bacillus subtilis , sporulation requires that the 26-amino acid protein SpoVM embeds specifically into the forespore membrane, a structure with convex curvature. How this nanometer-sized protein can detect curves on a micrometer scale is not well understood. Here, we report that SpoVM exploits a "dash-and-recruit" mechanism to preferentially accumulate on the forespore. Using time-resolved imaging and flow cytometry, we observe that SpoVM exhibits a faster adsorption rate onto membranes of higher convex curvature. This preferential adsorption is accurately modeled as a two-step process: first, an initial binding event occurs with a faster on rate, then cooperative recruitment of additional SpoVM molecules follows. We demonstrate that both this biochemical process and effective sporulation in vivo require an unstructured and flexible SpoVM N terminus. We propose that this two-pronged strategy of fast adsorption followed by recruitment of subsequent molecules is a general mechanism that allows small proteins to detect subtle curves with a radius 1,000-fold their size.

Spores of Bacillus subtilis are dormant cell types that are formed when the bacterium encounters starvation conditions. Spores are encased in a shell, termed the coat, which is composed of approximately seventy different proteins and protects the spore’s genetic material from environmental insults. The structural component of the basement layer of the coat is an exceptional cytoskeletal protein, termed SpoIVA, which binds and hydrolyzes ATP. ATP hydrolysis is utilized to drive a conformational change in SpoIVA that leads to its irreversible self-assembly into a static polymer in vitro. Here, we characterize the middle domain of SpoIVA, the predicted secondary structure of which resembles the chemotaxis protein CheX but, unlike CheX, does not harbor residues required for phosphatase activity. Disruptions in this domain did not abolish ATP hydrolysis, but resulted in mislocalization of the protein and reduction in sporulation efficiency in vivo. In vitro, disruptions in this domain prevented the ATP hydrolysis-driven conformational change in SpoIVA required for polymerization and led to the aggregation of SpoIVA into particles that did not form filaments. We propose a model in which SpoIVA initially assumes a conformation in which it inhibits its own aggregation into particles, and that ATP hydrolysis remodels the protein so that it assumes a polymerization-competent conformation.

Summary Although prokaryotes ordinarily undergo binary fission to produce two identical daughter cells, some are able to undergo alternative developmental pathways that produce daughter cells of distinct cell morphology and fate. One such example is a developmental programme called sporulation in the bacterium Bacillus subtilis, which occurs under conditions of environmental stress. Sporulation has long been used as a model system to help elucidate basic processes of developmental biology including transcription regulation, intercellular signalling, membrane remodelling, protein localization and cell fate determination. This review highlights some of the recent work that has been done to further understand prokaryotic cell differentiation during sporulation and its potential applications.

To characterise and correlate crystalline precipitations implicated in non-healing corneal ulceration in two patients with a previous history of acanthamoeba keratitis. Persistence of acanthamoeba and secondary bacterial infection was excluded with negative corneal scrapes. Confocal microscopy identified crystal-like deposits within the corneal stroma. To investigate possible precipitating combinations, all concurrent treatments at the time of presentation were mixed in wells, with observation of precipitate formation. Precipitates were observed with phase-contrast microscopy, and subsequently characterised via crystallography techniques and electrospray ionisation mass spectrometry. Combinations of dexamethasone 0.1% minims and chlorhexidine gluconate 0.2% formed an amorphous material characterised by electrospray ionisation mass spectrometry as an insoluble chlorhexidine salt. Combinations of chloramphenicol drops and timolol 0.5% formed a crystal identified via X-ray crystallography as santite (K(B5O6(OH)4).(H2O)2). This is a borate mineral identified in nature, arising from thermal springs, but never reported in biological tissues. Clinical improvement was observed following the cessation of the implicated precipitating combinations. Our observations suggest iatrogenic precipitate formation, with a potential deleterious effect upon healing. The substrates for these precipitates include several frequently prescribed topical ophthalmic treatments. These findings shed new light on the aetiopathogenesis of non-healing corneal ulceration, and have broad implications on topical prescribing for this challenging condition.

Biofilm formation in Bacillus subtilis requires the differentiation of a subpopulation of cells responsible for the production of the extracellular matrix that structures the biofilm. Differentiation of matrix-producing cells depends, among other factors, on the FloT and YqfA proteins. These proteins are present exclusively in functional membrane microdomains of B. subtilis and are homologous to the eukaryotic lipid raft-specific flotillin proteins. In the absence of FloT and YqfA, diverse proteins normally localized to the membrane microdomains of B. subtilis are not functional. Here we show that the absence of FloT and YqfA reduces the level of the septal-localized protease FtsH. The flotillin homologues FloT and YqfA are occasionally present at the midcell in exponentially growing cells and the absence of FloT and YqfA negatively affects FtsH concentration. Biochemical experiments indicate a direct interaction between FloT/YqfA and FtsH. Moreover, FtsH is essential for the differentiation of matrix producers and hence, biofilm formation. This molecular trigger of biofilm formation may therefore be used as a target for the design of new biofilm inhibitors. Accordingly, we show that the small protein SpoVM, known to bind to and inhibit FtsH activity, inhibits biofilm formation in B. subtilis and other distantly related bacteria.

Summary Mature spores of the bacterium Bacillus subtilis are encased by two concentric shells: an inner shell (the ‘cortex’), made of peptidoglycan; and an outer proteinaceous shell (the ‘coat’), whose basement layer is anchored to the surface of the developing spore via a 26-amino-acid-long protein called SpoVM. During sporulation, initiation of cortex assembly depends on the successful initiation of coat assembly, but the mechanisms that co-ordinate the morphogenesis of both structures are largely unknown. Here, we describe a sporulation pathway involving SpoVM and a 37-amino-acid-long protein named ‘CmpA’ that is encoded by a previously un-annotated gene and is expressed under control of two sporulation-specific transcription factors (σE and SpoIIID). CmpA localized to the surface of the developing spore and deletion of cmpA resulted in cells progressing through the sporulation programme more quickly. Overproduction of CmpA did not affect normal growth or cell division, but delayed entry into sporulation and abrogated cortex assembly. In those cells that had successfully initiated coat assembly, CmpA was removed by a post-translational mechanism, presumably in order to overcome the sporulation inhibition it imposed. We propose a model in which CmpA participates in a developmental checkpoint that ensures the proper orchestration of coat and cortex morphogenesis by repressing cortex assembly until coat assembly successfully initiates.

Purpose. To report the visual outcome of penetrating keratoplasty performed on the sympathizing eye in three cases of sympathetic ophthalmitis.Methods. Interventional case series of three patients, diagnosed with sympathetic ophthalmitis, with corneal changes in the form of band keratopathy and decompensation underwent penetrating keratoplasty to the sympathizing eye. They had each sustained penetrating trauma as a child and had undergone previous cataract surgery and superficial keratectomy. Two patients had undergone lamellar keratoplasty prior to this procedure. One patient had undergone trabeculectomy for glaucoma, and she was on antiglaucoma medication. The preoperative visual acuity was 1/60 in the affected eye of each patient. Penetrating keratoplasty was performed in the sympathizing eye and the donor graft size was 7.50 mm, and the host graft size was 7.25 mm. Our patients were immunosuppressed prior to the procedure to help prevent graft rejection.Result. At one year follow-up, a BCVA of 6/36 or better was achieved in all three patients. Postoperative examination of the fundus showed peripheral chorioretinal atrophy with pigmentary changes at the macula, accounting for the limited vision. The grafts remain clear to date, and there has been no recurrence of uveitis or rejection.Conclusion. Penetrating keratoplasty can be considered as a surgical option to restore useful vision in a stable sympathizing eye in sympathetic ophthalmitis, and this depends on the extent of the pathology. However, these cases require treatment with immunosuppressives to prevent graft rejection and to prolong graft survival.

Understanding the mechanisms that underlie the organization of bacterial cells has become a significant challenge in the field of bacterial cytology. Of specific interest are early macromolecular sorting events that establish cellular non-uniformity and provide chemical landmarks for later localization events. In this review, we will examine specific examples of lipids and proteins that appear to exploit differences in membrane curvature to drive their localization to particular regions of a bacterial cell. We will also discuss the physical limits of curvature-mediated localization within bacteria, and the use of modelling to infer biophysical properties of curvature-sensing macromolecules.

Proteins in bacteria often deploy to particular places within the cell, but the cues for localization are frequently mysterious. We found that the peripheral membrane protein SpoVM (VM) recognizes a geometric cue when localizing to a particular membrane during sporulation in Bacillus subtilis . Sporulation involves an inner cell maturing into a spore and an outer cell nurturing the developing spore. VM is produced in the outer cell, where it embeds in the membrane that surrounds the inner cell but not in the cytoplasmic membrane of the outer cell. We found that VM localized by discriminating between the positive curvature of the membrane surrounding the inner cell and the negative curvature of the cytoplasmic membrane. Membrane curvature could be a general cue for protein localization in bacteria.

A hallmark of morphogenesis is the orchestrated assembly of complex, supramolecular structures. One such structure is the proteineous coat that surrounds spores of the bacterium Bacillus subtilis. The coat is a multilayered shell that is composed of more than 50 proteins. These proteins assemble around a basement layer composed of the morphogenetic protein SpoIVA. We show that SpoIVA harbors a Walker A box that is required for the proper deployment of the protein to the surface of the developing spore and proper assembly of the entire coat. We further show that purified SpoIVA both binds and hydrolyzes ATP and that the protein self-assembles into cable-like structures in a manner that depends on ATP hydrolysis. Self-assembly driven by ATP is an unusual mechanism for the construction of a large cellular structure.

Aim: To report a preliminary study on the safety and efficacy of the use of a cheese-wire suture in trabeculectomy. Patients and methods: The case notes of 32 eyes of 25 patients with medically uncontrolled glaucoma who underwent trabeculectomy with cheese-wire suture at Stobhill Hospital, Glasgow, UK, between July 2001 and September 2002 were studied retrospectively. Diagnoses included primary open angle glaucoma (n = 24), normal tension glaucoma (n = 3), angle closure glaucoma (n = 2), ocular hypertension (n = 1), angle recession glaucoma (n = 1) and combined mechanism glaucoma (n = 1). The mean presenting intraocular pressure (IOP) was 29.5 mm Hg and mean intraocular pressure before operation was 23.5 mm Hg Outcome measures: Success was defined as lowering of IOP by at least 15% compared with IOP before removal. Results: A total of 20 eyes (62%) underwent removal of the cheese-wire suture. 17 eyes (85%) underwent removal for therapeutic reasons (failing/failed blebs) and three eyes (15%) underwent suture removal as the suture loops were exposed. The timing of removal was between 2 weeks and 12 months following surgery. A successful outcome was seen in 12/17 (70%) eyes in the eyes that had therapeutic suture removal. 15 eyes had undergone previous surgical interventions (trabeculectomy n = 12, extracapsular cataract extraction n = 1 and laser peripheral iridotomies n = 2). Mean IOP before removal was 23.66 mm Hg and mean IOP immediately following removal was 11.33 mm Hg. Of the 32 eyes that underwent trabeculectomy with cheese-wire suture, 24 eyes had intraoperative mitomycin C and one eye had 5-fluorouracil. The remaining seven eyes did not have any antimetabolites. Early complications related to the surgical procedure included conjunctival haematoma in one eye (3%), corneal abrasion in one eye (3%), wound leak in five eyes (15%), shallow anterior chamber in one eye (3%), hyphaema in six eyes (18%), choroidal effusion in six eyes (18%) and raised IOP in two eyes (6%). Late complications of suture exposure occurred in three eyes (9%). Complications related to removal of the cheese-wire suture included suture breakage in two eyes (10%), hypotony in one eye (5%) and transient hyphaema in one eye (5%). Conclusion: The use of cheese-wire suture in trabeculectomy appears to be safe and may provide an alternative strategy in the management of bleb failure.

SummaryRecent discoveries of regulated cell death in bacteria have led to speculation about possible benefits that apoptosis-like pathways may confer to single-celled organisms. However, establishing how these pathways provide increased ecological fitness has remained difficult to determine. Here, we report a pathway in Bacillus subtilis in which regulated cell death maintains the fidelity of sporulation through selective removal of cells that misassemble the spore envelope. The spore envelope, which protects the dormant spore’s genome from environmental insults, uses the protein SpoIVA as a scaffold for assembly. We found that disrupting envelope assembly activates a cell death pathway wherein the small protein CmpA acts as an adaptor to the AAA+ ClpXP protease to degrade SpoIVA, thereby halting sporulation and resulting in lysis of defective sporulating cells. We propose that removal of unfit cells from a population of terminally differentiating cells protects against evolutionary deterioration and ultimately loss of the sporulation program.

Dormant bacterial spores are encased in a thick protein shell, the ‘coat', which contains ∼70 different proteins. The coat protects the spore from environmental insults, and is among the most durable static structures in biology. Owing to extensive cross-linking among coat proteins, this structure has been recalcitrant to detailed biochemical analysis, so molecular details of how it assembles are largely unknown. Here, we reconstitute the basement layer of the coat atop spherical membranes supported by silica beads to create artificial spore-like particles. We report that these synthetic spore husk-encased lipid bilayers (SSHELs) assemble and polymerize into a static structure, mimicking in vivo basement layer assembly during sporulation in Bacillus subtilis. In addition, we demonstrate that SSHELs may be easily covalently modified with small molecules and proteins. We propose that SSHELs may be versatile display platforms for drugs and vaccines in clinical settings, or for enzymes that neutralize pollutants for environmental remediation., The densely crosslinked protein coats of bacterial spores are among the most durable static structures in biology. Wu et al. reconstitute the basement layer of a bacterial spore coat on membrane-coated beads, and generate covalently-modified spore-like particles with therapeutic potential.

In bacteria, certain shape-sensing proteins localize to differently curved membranes. During sporulation in Bacillus subtilis, the only convex (positively curved) surface in the cell is the forespore, an approximately spherical internal organelle. Previously, we demonstrated that SpoVM localizes to the forespore by preferentially adsorbing onto slightly convex membranes. Here, we used NMR and molecular dynamics simulations of SpoVM and a localization mutant (SpoVM(P9A)) to reveal that SpoVM's atypical amphipathic α-helix inserts deeply into the membrane and interacts extensively with acyl chains to sense packing differences in differently curved membranes. Based on binding to spherical supported lipid bilayers and Monte Carlo simulations, we hypothesize that SpoVM's membrane insertion, along with potential cooperative interactions with other SpoVM molecules in the lipid bilayer, drives its preferential localization onto slightly convex membranes. Such a mechanism, which is distinct from that used by high curvature-sensing proteins, may be widely conserved for the localization of proteins onto the surface of cellular organelles.

Summary Spore formation in Bacillus subtilis involves the formation of a thick, proteinaceous shell or coat that is assembled around a specialized membrane known as the outer forespore membrane. Here we present evidence that the assembling coat is tethered to the outer forespore membrane by a 26-amino-acid peptide called SpoVM, which is believed to form an amphipathic helix. We show that proper localization of SpoVM is dependent on SpoIVA, a morphogenetic protein that forms the basement layer of the spore coat, and conversely, that proper localization of SpoIVA is dependent on SpoVM. Genetic, biochemical and cytological evidence indicates that this mutual dependence is mediated in part by contact between an amino acid side-chain located near the extreme C-terminus of SpoIVA and an amino acid side-chain on the hydrophilic face of the SpoVM helix. Evidence is also presented that SpoVM adheres to the outer forespore membrane via hydrophobic, amino acid side-chains on the hydrophobic face of the helix. The results suggest that the SpoVM helix is oriented parallel to the membrane with the hydrophobic face buried in the lipid bilayer.

Yersinia spp. inject virulence proteins called Yops into the cytosol of target eukaryotic cells in an effort to evade phagocytic killing via a dedicated protein-sorting pathway termed type III secretion. Previous studies have proposed that, unlike other protein translocation mechanisms, Yops are not recognized as substrates for secretion via a solely proteinaceous signal. Rather, at least some of this information may be encoded within yop mRNA. Herein, we report that the first seven codons of yopE , when fused to the reporter protein neomycin phosphotransferase (Npt), are sufficient for the secretion of YopE 1-7 -Npt when type III secretion is induced in vitro. Systematic mutagenesis of yopE codons 1 to 7 reveals that, like yopQ , codons 2, 3, 5, and 7 are sensitive to mutagenesis, thereby defining the first empirical similarity between the secretion signals of two type III secreted substrates. Like that of yopQ , the secretion signal of yopE exhibits a bipartite nature. This is manifested by the ability of codons 8 to 15 to suppress point mutations in the minimal secretion signal that change the amino acid specificities of particular codons or that induce alterations in the reading frame. Further, we have identified a single nucleotide position in codon 3 that, when mutated, conserves the predicted amino acid sequence of the YopE 1-7 -Npt but abrogates secretion of the reporter protein. When introduced into the context of the full-length yopE gene, the single-nucleotide mutation reduces the type III injection of YopE into HeLa cells, even though the predicted amino acid sequence remains the same. Thus, yopE mRNA appears to encode a property that mediates the type III injection of YopE.

The type III secretion signal of Yersinia enterocolitica YopN was mapped using a gene fusion approach. yopN codons 1 to 12 were identified as critical for signal function. Several synonymous mutations that abolish secretion of hybrid proteins without altering the codon specificity of yopN mRNA were identified.

Type III secretion is the designation given to those protein secretion pathways, primarily in pathogenic Gram-negative bacteria, whose secretion machinery components share an amino acid sequence homology to components of the flagellar basal body. In Yersinia spp., these secretion machineries inject virulence proteins called Yops into the cytosol of target macrophages in an effort to evade phagocytic killing. To date, a clear mechanism by which Yops are recognized by the type III secretion machinery has not been elucidated. Unlike most, if not all, previously characterized protein sorting pathways, the information that identifies Yops as substrates for secretion seems not to be wholly encoded within the Yop peptide sequence. In fact, it appears that at least some of this information is contained within yop mRNAs. This review summarizes recent observations that have been made in this unusual field and proposes models by which proteins may be initiated into this pathway.

The type III machinery of Yersinia transports Yop proteins across the bacterial envelope. The minimal secretion signal of yopQ is located in codons 1-10 that, when fused in frame to the neomycin phosphotransferase gene, is sufficient to promote type III secretion of YopQ 1 - 1 0 -Npt. Frame-shift mutations, generated by nucleotide insertions or deletions following the AUG start and suppressed at the fusion site with npt, abrogate signalling of yopQ 1 - 1 0 but not of yopQ 1 - 1 5 . By generating transversions of every single nucleotide in yopQ 1 - 1 0 , we identified 10 nucleotide positions in codons 2, 3, 5, 7, 9 and 10 that were each required for substrate recognition. One transversion that abolishes secretion, uridyl 9 to adenyl (U9A), is a synonymous codon 3 mutation that retains the original amino acid as confirmed by Edman degradation analysis, suggesting that the mRNA but not the amino acid sequence of yopQ 1 - 1 0 is involved in secretion signalling. Although transversion of U8A abrogates signalling of yopQ 1 - 1 0 , fusion of yopO codons 11-15 restores secretion. The nucleotides that are required for this suppression by yopQ 1 1 - 1 5 were identified and revealed both synonymous and non-synonymous mutations. Frame-shift mutations introduced into just this suppressor region (codons 11-15) did not abrogate its ability to suppress mutations in the minimal secretion signal (codons 1-10). Thus, elements downstream of the minimal secretion signal of YopQ increase the efficiency of YopQ secretion and suppress mutations elsewhere in the secretion signal.

Sporulation in the bacterium Bacillus subtilis is a developmental program in which a progenitor cell differentiates into two different cell types, the smaller of which eventually becomes a dormant cell called a spore. The process begins with an asymmetric cell division event, followed by the activation of a transcription factor, σF, specifically in the smaller cell. Here, we show that the structural protein DivIVA localizes to the polar septum during sporulation and is required for asymmetric division and the compartment-specific activation of σF. Both events are known to require a protein called SpoIIE, which also localizes to the polar septum. We show that DivIVA copurifies with SpoIIE and that DivIVA may anchor SpoIIE briefly to the assembling polar septum before SpoIIE is subsequently released into the forespore membrane and recaptured at the polar septum. Finally, using super-resolution microscopy, we demonstrate that DivIVA and SpoIIE ultimately display a biased localization on the side of the polar septum that faces the smaller compartment in which σF is activated., Author Summary A central feature of developmental programs is the establishment of asymmetry and the production of genetically identical daughter cells that display different cell fates. Sporulation in the bacterium Bacillus subtilis is a simple developmental program in which the cell divides asymmetrically to produce two daughter cells, after which the transcription factor σF is activated specifically in the smaller cell. Here we investigated DivIVA, which localizes to highly negatively curved membranes, and discovered that it localizes at the asymmetric division site. In the absence of DivIVA, cells failed to asymmetrically divide and prematurely activated σF in the predivisional cell, largely unreported phenotypes for any deletion mutant in a sporulation gene. We found that DivIVA copurifies with SpoIIE, a protein that is required for asymmetric division and σF activation, and that both proteins preferentially localize on the side of the septum facing the smaller daughter cell. DivIVA is therefore a previously overlooked structural factor that is required at the onset of sporulation to mediate both asymmetric division and compartment-specific transcription.

Small proteins, here defined as proteins of 50 amino acids or fewer in the absence of processing, have traditionally been overlooked due to challenges in their annotation and biochemical detection. In the past several years, however, increasing numbers of small proteins have been identified either through the realization that mutations in intergenic regions are actually within unannotated small protein genes or through the discovery that some small, regulatory RNAs encode small proteins. These insights, together with comparative sequence analysis, indicate that tens if not hundreds of small proteins are synthesized in a given organism. This review summarizes what has been learned about the functions of several of these bacterial small proteins, most of which act at the membrane, illustrating the astonishing range of processes in which these small proteins act and suggesting several general conclusions. Important questions for future studies of these overlooked proteins are also discussed.

We describe the case of a 53-year-old woman with keratoconus who presented with a dislocated intrastromal corneal ring segment (ICRS) (Intacs) in the anterior chamber 3 weeks after surgery. To our knowledge, this is the first report of a late occurrence of postoperative anterior chamber dislocation of an ICRS and illustrates the need to consider the potential for such a complication. Financial Disclosure No author has a financial or proprietary interest in any material or method mentioned.

Thermosensors are ubiquitous integral membrane proteins found in all kinds of life. They are involved in many physiological roles, including membrane remodeling, chemotaxis, touch, and pain [1-3], but, the mechanism by which their transmembrane (TM) domains transmit temperature signals is largely unknown. The histidine kinase DesK from Bacillus subtilis is the paradigmatic example of a membrane-bound thermosensor suited to remodel membrane fluidity when the temperature drops below approximately 30°C [1, 4] providing, thus, a tractable system for investigating the mechanism of TM-mediated input-output control of thermal adaptation. Here we show that the multimembrane-spanning domain from DesK can be simplified into a chimerical single-membrane-spanning minimal sensor (MS) that fully retains, in vivo and in vitro, the sensing properties of the parental system. The MS N terminus contains three hydrophilic amino acids near the lipid-water interface creating an instability hot spot. Mutational analysis of this boundary-sensitive beacon revealed that membrane thickness controls the signaling state of the sensor by dictating the hydration level of the metastable hydrophilic spot. Guided by these results we biochemically demonstrated that the MS signal transmission activity is sensitive to bilayer thickness. Membrane thickness could be a general cue for sensing temperature in many organisms.