Detection of membrane fission in single Bacillus subtilis cells during endospore formation with high temporal resolution.

Membrane fission is an essential process in all domains of life. The underlying mechanisms remain poorly understood in bacteria, partly because suitable assays are lacking. Here, we describe an assay to detect membrane fission during endospore formation in single Bacillus subtilis cells with a temporal resolution of ∼1 min. Other cellular processes can be quantified and temporally aligned to the membrane fission event in individual cells, revealing correlations and causal relationships. For complete details on the use and execution of this protocol, please refer to Landajuela et al.1.

Ultrastructure of macromolecular assemblies contributing to bacterial spore resistance revealed by in situ cryo-electron tomography.

Bacterial spores owe their incredible resistance capacities to molecular structures that protect the cell content from external aggressions. Among the determinants of resistance are the quaternary structure of the chromosome and an extracellular shell made of proteinaceous layers (the coat), the assembly of which remains poorly understood. Here, in situ cryo-electron tomography on lamellae generated by cryo-focused ion beam micromachining provides insights into the ultrastructural organization of Bacillus subtilis sporangia. The reconstructed tomograms reveal that early during sporulation, the chromosome in the forespore adopts a toroidal structure harboring 5.5-nm thick fibers. At the same stage, coat proteins at the surface of the forespore form a stack of amorphous or structured layers with distinct electron density, dimensions and organization. By analyzing mutant strains using cryo-electron tomography and transmission electron microscopy on resin sections, we distinguish seven nascent coat regions with different molecular properties, and propose a model for the contribution of coat morphogenetic proteins.

Genetic Screens Identify Additional Genes Implicated in Envelope Remodeling during the Engulfment Stage of Bacillus subtilis Sporulation.

During bacterial endospore formation, the developing spore is internalized into the mother cell through a phagocytic-like process called engulfment, which involves synthesis and hydrolysis of peptidoglycan. Engulfment peptidoglycan hydrolysis requires the widely conserved and well-characterized DMP complex, composed of SpoIID, SpoIIM, and SpoIIP. In contrast, although peptidoglycan synthesis has been implicated in engulfment, the protein players involved are less well defined. The widely conserved SpoIIIAH-SpoIIQ interaction is also required for engulfment efficiency, functioning like a ratchet to promote membrane migration around the forespore. Here, we screened for additional factors required for engulfment using transposon sequencing in Bacillus subtilis mutants with mild engulfment defects. We discovered that YrvJ, a peptidoglycan hydrolase, and the MurA paralog MurAB, involved in peptidoglycan precursor synthesis, are required for efficient engulfment. Cytological analyses suggest that both factors are important for engulfment when the DMP complex is compromised and that MurAB is additionally required when the SpoIIIAH-SpoIIQ ratchet is abolished. Interestingly, despite the importance of MurAB for sporulation in B. subtilis, phylogenetic analyses of MurA paralogs indicate that there is no correlation between sporulation and the number of MurA paralogs and further reveal the existence of a third MurA paralog, MurAC, within the Firmicutes. Collectively, our studies identify two new factors that are required for efficient envelop remodeling during sporulation and highlight the importance of peptidoglycan precursor synthesis for efficient engulfment in B. subtilis and likely other endospore-forming bacteria. IMPORTANCE In bacteria, cell envelope remodeling is critical for cell growth and division. This is also the case during the development of bacteria into highly resistant endospores (spores), known as sporulation. During sporulation, the developing spore becomes internalized inside the mother cell through a phagocytic-like process called engulfment, which is essential to form the cell envelope of the spore. Engulfment involves both the synthesis and hydrolysis of peptidoglycan and the stabilization of migrating membranes around the developing spore. Importantly, although peptidoglycan synthesis has been implicated during engulfment, the specific genes that contribute to this molecular element of engulfment have remained unclear. Our study identifies two new factors that are required for efficient envelope remodeling during engulfment and emphasizes the importance of peptidoglycan precursor synthesis for efficient engulfment in the model organism Bacillus subtilis and likely other endospore-forming bacteria. Finally, our work highlights the power of synthetic screens to reveal additional genes that contribute to essential processes during sporulation.

Membrane fission during bacterial spore development requires cellular inflation driven by DNA translocation.

Bacteria require membrane fission for both cell division and endospore formation. In Bacillus subtilis, sporulation initiates with an asymmetric division that generates a large mother cell and a smaller forespore that contains only a quarter of its genome. As the mother cell membranes engulf the forespore, a DNA translocase pumps the rest of the chromosome into the small forespore compartment, inflating it due to increased turgor. When the engulfing membrane undergoes fission, the forespore is released into the mother cell cytoplasm. The B. subtilis protein FisB catalyzes membrane fission during sporulation, but the molecular basis is unclear. Here, we show that forespore inflation and FisB accumulation are both required for an efficient membrane fission. Forespore inflation leads to higher membrane tension in the engulfment membrane than in the mother cell membrane, causing the membrane to flow through the neck connecting the two membrane compartments. Thus, the mother cell supplies some of the membrane required for the growth of the membranes surrounding the forespore. The oligomerization of FisB at the membrane neck slows the equilibration of membrane tension by impeding the membrane flow. This leads to a further increase in the tension of the engulfment membrane, promoting its fission through lysis. Collectively, our data indicate that DNA translocation has a previously unappreciated second function in energizing the FisB-mediated membrane fission under energy-limited conditions.

Hydroxyl Radical Overproduction in the Envelope: an Achilles' Heel in Peptidoglycan Synthesis.

While many mechanisms governing bacterial envelope homeostasis have been identified, others remain poorly understood. To decipher these processes, we previously developed an assay in the Gram-negative model Escherichia coli to identify genes involved in maintenance of envelope integrity. One such gene was ElyC, which was shown to be required for envelope integrity and peptidoglycan synthesis at room temperature. ElyC is predicted to be an integral inner membrane protein with a highly conserved domain of unknown function (DUF218). In this study, and stemming from a further characterization of the role of ElyC in maintaining cell envelope integrity, we serendipitously discovered an unappreciated form of oxidative stress in the bacterial envelope. We found that cells lacking ElyC overproduce hydroxyl radicals (HO•) in their envelope compartment and that HO• overproduction is directly or indirectly responsible for the peptidoglycan synthesis arrest, cell envelope integrity defects, and cell lysis of the ΔelyC mutant. Consistent with these observations, we show that the ΔelyC mutant defect is suppressed during anaerobiosis. HO• is known to cause DNA damage but to our knowledge has not been shown to interfere with peptidoglycan synthesis. Thus, our work implicates oxidative stress as an important stressor in the bacterial cell envelope and opens the door to future studies deciphering the mechanisms that render peptidoglycan synthesis sensitive to oxidative stress. IMPORTANCE Oxidative stress is caused by the production and excessive accumulation of oxygen reactive species. In bacterial cells, oxidative stress mediated by hydroxyl radicals is typically associated with DNA damage in the cytoplasm. Here, we reveal the existence of a pathway for oxidative stress in the envelope of Gram-negative bacteria. Stemming from the characterization of a poorly characterized gene, we found that HO• overproduction specifically in the envelope compartment causes inhibition of peptidoglycan synthesis and eventually bacterial cell lysis.

FtsK and SpoIIIE, coordinators of chromosome segregation and envelope remodeling in bacteria.

The translocation of DNA during bacterial cytokinesis is mediated by the SpoIIIE/FtsK family of proteins. These proteins ensure efficient chromosome segregation into sister cells by ATP-driven translocation of DNA and they control chromosome dimer resolution. How FtsK/SpoIIIE mediate chromosome translocation during cytokinesis in Gram-positive and Gram-negative organisms has been the subject of debate. Studies on FtsK in Escherichia coli, and recent work on SpoIIIE in Bacillus subtilis, have identified interactions between each translocase and the division machinery, supporting the idea that SpoIIIE and FtsK coordinate the final steps of cytokinesis with completion of chromosome segregation. Here we summarize and discuss the view that SpoIIIE and FtsK play similar roles in coordinating cytokinesis with chromosome segregation, during growth and differentiation.

Structural insights into ring-building motif domains involved in bacterial sporulation.

Components of specialized secretion systems, which span the inner and outer membranes in Gram-negative bacteria, include ring-forming proteins whose oligomerization was proposed to be promoted by domains called RBM for "Ring-Building Motifs". During spore formation in Gram-positive bacteria, a transport system called the SpoIIIA-SpoIIQ complex also assembles in the double membrane that surrounds the forespore following its endocytosis by the mother cell. The presence of RBM domains in some of the SpoIIIA proteins led to the hypothesis that they would assemble into rings connecting the two membranes and form a conduit between the mother cell and forespore. Among them, SpoIIIAG forms homo-oligomeric rings in vitro but the oligomerization of other RBM-containing SpoIIIA proteins, including SpoIIIAH, remains to be demonstrated. In this work, we identified RBM domains in the YhcN/YlaJ family of proteins that are not related to the SpoIIIA-SpoIIQ complex. We solved the crystal structure of YhcN from Bacillus subtilis, which confirmed the presence of a RBM fold, flanked by additional secondary structures. As the protein did not show any oligomerization ability in vitro, we investigated the structural determinants of ring formation in SpoIIIAG, SpoIIIAH and YhcN. We showed that in vitro, the conserved core of RBM domains alone is not sufficient for oligomerization while the ß-barrel forming region in SpoIIIAG forms rings on its own. This work suggests that some RBMs might indeed participate in the assembly of homomeric rings but others might have evolved toward other functions.

FisB relies on homo-oligomerization and lipid binding to catalyze membrane fission in bacteria.

Little is known about mechanisms of membrane fission in bacteria despite their requirement for cytokinesis. The only known dedicated membrane fission machinery in bacteria, fission protein B (FisB), is expressed during sporulation in Bacillus subtilis and is required to release the developing spore into the mother cell cytoplasm. Here, we characterized the requirements for FisB-mediated membrane fission. FisB forms mobile clusters of approximately 12 molecules that give way to an immobile cluster at the engulfment pole containing approximately 40 proteins at the time of membrane fission. Analysis of FisB mutants revealed that binding to acidic lipids and homo-oligomerization are both critical for targeting FisB to the engulfment pole and membrane fission. Experiments using artificial membranes and filamentous cells suggest that FisB does not have an intrinsic ability to sense or induce membrane curvature but can bridge membranes. Finally, modeling suggests that homo-oligomerization and trans-interactions with membranes are sufficient to explain FisB accumulation at the membrane neck that connects the engulfment membrane to the rest of the mother cell membrane during late stages of engulfment. Together, our results show that FisB is a robust and unusual membrane fission protein that relies on homo-oligomerization, lipid binding, and the unique membrane topology generated during engulfment for localization and membrane scission, but surprisingly, not on lipid microdomains, negative-curvature lipids, or curvature sensing.

Pitfalls in the assessment of disseminated intravascular coagulation in patients on dabigatran.

Dabigatran is an orally administrated anticoagulant that directly inhibits thrombin. However, the drug can affect routine coagulation tests such as prothrombin time (PT), activated partial thromboplastin time (APTT) and thrombin time (TT), as well as haemostasis assays, (e.g., clot-based coagulation factor assays). There are limited data on the effect of dabigatran on some fibrinogen measurements and on D-dimer assays, both important components in the laboratory assessment of disseminated intravascular coagulation (DIC). The objectives of this study were: (1) to determine the effects of various concentrations of dabigatran on fibrinogen and D-Dimer assays; and (2) to compare the von Clauss method of fibrinogen measurement using two reagents with differing thrombin concentrations (35 UNIH/mL and 100 UNIH/mL) and PT-derived fibrinogen measurement in the presence of the drug. Aliquots of pooled normal plasma were spiked with different concentrations of dabigatran to reflect in vivo on-therapy levels as well as levels observed in cases of massive accumulation of the drug. Of the routine coagulation assays, in ascending order of sensitivity to dabigatran were PT, APTT and TT. The von Clauss method of measuring fibrinogen using a reagent with low thrombin concentration was affected even at drug levels corresponding to in vivo trough concentrations, whereas the reagent with higher thrombin concentration was only affected at drug levels that were above observed peak concentrations in patients taking 150 mg of the drug twice daily. PT-derived fibrinogen was affected at approximately in vivo peak drug concentrations. The D-dimer assay was affected only at drug concentrations well above peak drug levels. Attempts at in vitro neutralisation of the drug with DOAC-Stop resulted in 'correction' of some of these measurements depending on drug concentration. Like the routine coagulation assays, there is a dabigatran concentration dependent effect on the accuracy of fibrinogen and D-dimer assays. Falsely low fibrinogen results due to dabigatran may confound the assessment of DIC and diagnostic laboratories need to evaluate the performance of their own reagents.

Chromosome Segregation and Peptidoglycan Remodeling Are Coordinated at a Highly Stabilized Septal Pore to Maintain Bacterial Spore Development.

Asymmetric division, a hallmark of endospore development, generates two cells, a larger mother cell and a smaller forespore. Approximately 75% of the forespore chromosome must be translocated across the division septum into the forespore by the DNA translocase SpoIIIE. Asymmetric division also triggers cell-specific transcription, which initiates septal peptidoglycan remodeling involving synthetic and hydrolytic enzymes. How these processes are coordinated has remained a mystery. Using Bacillus subtilis, we identified factors that revealed the link between chromosome translocation and peptidoglycan remodeling. In cells lacking these factors, the asymmetric septum retracts, resulting in forespore cytoplasmic leakage and loss of DNA translocation. Importantly, these phenotypes depend on septal peptidoglycan hydrolysis. Our data support a model in which SpoIIIE is anchored at the edge of a septal pore, stabilized by newly synthesized peptidoglycan and protein-protein interactions across the septum. Together, these factors ensure coordination between chromosome translocation and septal peptidoglycan remodeling to maintain spore development.

A dynamic, ring-forming MucB / RseB-like protein influences spore shape in Bacillus subtilis.

How organisms develop into specific shapes is a central question in biology. The maintenance of bacterial shape is connected to the assembly and remodelling of the cell envelope. In endospore-forming bacteria, the pre-spore compartment (the forespore) undergoes morphological changes that result in a spore of defined shape, with a complex, multi-layered cell envelope. However, the mechanisms that govern spore shape remain poorly understood. Here, using a combination of fluorescence microscopy, quantitative image analysis, molecular genetics and transmission electron microscopy, we show that SsdC (formerly YdcC), a poorly-characterized new member of the MucB / RseB family of proteins that bind lipopolysaccharide in diderm bacteria, influences spore shape in the monoderm Bacillus subtilis. Sporulating cells lacking SsdC fail to adopt the typical oblong shape of wild-type forespores and are instead rounder. 2D and 3D-fluorescence microscopy suggest that SsdC forms a discontinuous, dynamic ring-like structure in the peripheral membrane of the mother cell, near the mother cell proximal pole of the forespore. A synthetic sporulation screen identified genetic relationships between ssdC and genes involved in the assembly of the spore coat. Phenotypic characterization of these mutants revealed that spore shape, and SsdC localization, depend on the coat basement layer proteins SpoVM and SpoIVA, the encasement protein SpoVID and the inner coat protein SafA. Importantly, we found that the ΔssdC mutant produces spores with an abnormal-looking cortex, and abolishing cortex synthesis in the mutant largely suppresses its shape defects. Thus, SsdC appears to play a role in the proper assembly of the spore cortex, through connections to the spore coat. Collectively, our data suggest functional diversification of the MucB / RseB protein domain between diderm and monoderm bacteria and identify SsdC as an important factor in spore shape development.

The ParB homologs, Spo0J and Noc, together prevent premature midcell Z ring assembly when the early stages of replication are blocked in Bacillus subtilis.

Precise cell division in coordination with DNA replication and segregation is of utmost importance for all organisms. The earliest stage of cell division is the assembly of a division protein FtsZ into a ring, known as the Z ring, at midcell. What still eludes us, however, is how bacteria precisely position the Z ring at midcell. Work in B. subtilis over the last two decades has identified a link between the early stages of DNA replication and cell division. A recent model proposed that the progression of the early stages of DNA replication leads to an increased ability for the Z ring to form at midcell. This model arose through studies examining Z ring position in mutants blocked at different steps of the early stages of DNA replication. Here, we show that this model is unlikely to be correct and the mutants previously studied generate nucleoids with different capacity for blocking midcell Z ring assembly. Importantly, our data suggest that two proteins of the widespread ParB family, Noc and Spo0J are required to prevent Z ring assembly over the bacterial nucleoid and help fine tune the assembly of the Z ring at midcell during the cell cycle.

Evidence that regulation of intramembrane proteolysis is mediated by substrate gating during sporulation in Bacillus subtilis.

During the morphological process of sporulation in Bacillus subtilis two adjacent daughter cells (called the mother cell and forespore) follow different programs of gene expression that are linked to each other by signal transduction pathways. At a late stage in development, a signaling pathway emanating from the forespore triggers the proteolytic activation of the mother cell transcription factor σK. Cleavage of pro-σK to its mature and active form is catalyzed by the intramembrane cleaving metalloprotease SpoIVFB (B), a Site-2 Protease (S2P) family member. B is held inactive by two mother-cell membrane proteins SpoIVFA (A) and BofA. Activation of pro-σK processing requires a site-1 signaling protease SpoIVB (IVB) that is secreted from the forespore into the space between the two cells. IVB cleaves the extracellular domain of A but how this cleavage activates intramembrane proteolysis has remained unclear. Structural studies of the Methanocaldococcus jannaschii S2P homolog identified closed (substrate-occluded) and open (substrate-accessible) conformations of the protease, but the biological relevance of these conformations has not been established. Here, using co-immunoprecipitation and fluorescence microscopy, we show that stable association between the membrane-embedded protease and its substrate requires IVB signaling. We further show that the cytoplasmic cystathionine-ß-synthase (CBS) domain of the B protease is not critical for this interaction or for pro-σK processing, suggesting the IVB-dependent interaction site is in the membrane protease domain. Finally, we provide evidence that the B protease domain adopts both open and closed conformations in vivo. Collectively, our data support a substrate-gating model in which IVB-dependent cleavage of A on one side of the membrane triggers a conformational change in the membrane-embedded protease from a closed to an open state allowing pro-σK access to the caged interior of the protease.

Structural characterization of the sporulation protein GerM from Bacillus subtilis.

The Gram-positive bacterium Bacillus subtilis responds to starvation by entering a morphological differentiation process leading to the formation of a highly resistant spore. Early in the sporulation process, the cell asymmetrically divides into a large compartment (the mother cell) and a smaller one (the forespore), which will maturate into a resistant spore. Proper development of the forespore requires the assembly of a multiprotein complex called the SpoIIIA-SpoIIQ complex or "A-Q complex". This complex involves the forespore protein SpoIIQ and eight mother cell proteins (SpoIIIAA to SpoIIIAH), many of which share structural similarities with components of specialized secretion systems and flagella found in Gram-negative bacteria. The assembly of the A-Q complex across the two membranes that separate the mother cell and forespore was recently shown to require GerM. GerM is a lipoprotein composed of two GerMN domains, a family of domains with unknown function. Here, we report X-ray crystallographic structures of the first GerMN domain of GerM at 1.0 Šresolution, and of the soluble domain of GerM (the tandem of GerMN domains) at 2.1 Šresolution. These structures reveal that GerMN domains can adopt distinct conformations and that the core of these domains display structural similarities with ring-building motifs found in components of specialized secretion system and in SpoIIIA proteins. This work provides an additional piece towards the structural characterization of the A-Q complex.

The New Kid on the Block: A Specialized Secretion System during Bacterial Sporulation.

The transport of proteins across the bacterial cell envelope is mediated by protein complexes called specialized secretion systems. These nanomachines exist in both Gram-positive and Gram-negative bacteria and have been categorized into different types based on their structural components and function. Interestingly, multiple studies suggest the existence of a protein complex in endospore-forming bacteria that appears to be a new type of specialized secretion system. This protein complex is called the SpoIIIA-SpoIIQ complex and is an exception to the categorical norm since it appears to be a hybrid composed of different parts from well-defined specialized secretion systems. Here we summarize and discuss the current understanding of this complex and its potential role as a specialized secretion system.

A two-step transport pathway allows the mother cell to nurture the developing spore in Bacillus subtilis.

One of the hallmarks of bacterial endospore formation is the accumulation of high concentrations of pyridine-2,6-dicarboxylic acid (dipicolinic acid or DPA) in the developing spore. This small molecule comprises 5-15% of the dry weight of dormant spores and plays a central role in resistance to both wet heat and desiccation. DPA is synthesized in the mother cell at a late stage in sporulation and must be translocated across two membranes (the inner and outer forespore membranes) that separate the mother cell and forespore. The enzymes that synthesize DPA and the proteins required to translocate it across the inner forespore membrane were identified over two decades ago but the factors that transport DPA across the outer forespore membrane have remained mysterious. Here, we report that SpoVV (formerly YlbJ) is the missing DPA transporter. SpoVV is produced in the mother cell during the morphological process of engulfment and specifically localizes in the outer forespore membrane. Sporulating cells lacking SpoVV produce spores with low levels of DPA and cells engineered to express SpoVV and the DPA synthase during vegetative growth accumulate high levels of DPA in the culture medium. SpoVV resembles concentrative nucleoside transporters and mutagenesis of residues predicted to form the substrate-binding pocket supports the idea that SpoVV has a similar structure and could therefore function similarly. These findings provide a simple two-step transport mechanism by which the mother cell nurtures the developing spore. DPA produced in the mother cell is first translocated into the intermembrane space by SpoVV and is then imported into the forespore by the SpoVA complex. This pathway is likely to be broadly conserved as DPA synthase, SpoVV, and SpoVA proteins can be found in virtually all endospore forming bacteria.

The Bacillus subtilis germinant receptor GerA triggers premature germination in response to morphological defects during sporulation.

During sporulation in Bacillus subtilis, germinant receptors assemble in the inner membrane of the developing spore. In response to specific nutrients, these receptors trigger germination and outgrowth. In a transposon-sequencing screen, we serendipitously discovered that loss of function mutations in the gerA receptor partially suppress the phenotypes of > 25 sporulation mutants. Most of these mutants have modest defects in the assembly of the spore protective layers that are exacerbated in the presence of a functional GerA receptor. Several lines of evidence indicate that these mutants inappropriately trigger the activation of GerA during sporulation resulting in premature germination. These findings led us to discover that up to 8% of wild-type sporulating cells trigger premature germination during differentiation in a GerA-dependent manner. This phenomenon was observed in domesticated and undomesticated wild-type strains sporulating in liquid and on solid media. Our data indicate that the GerA receptor is poised on a knife's edge during spore development. We propose that this sensitized state ensures a rapid response to nutrient availability and also elicits premature germination of spores with improperly assembled protective layers resulting in the elimination of even mildly defective individuals from the population.

A ring-shaped conduit connects the mother cell and forespore during sporulation in Bacillus subtilis.

During spore formation in Bacillus subtilis a transenvelope complex is assembled across the double membrane that separates the mother cell and forespore. This complex (called the "A-Q complex") is required to maintain forespore development and is composed of proteins with remote homology to components of type II, III, and IV secretion systems found in Gram-negative bacteria. Here, we show that one of these proteins, SpoIIIAG, which has remote homology to ring-forming proteins found in type III secretion systems, assembles into an oligomeric ring in the periplasmic-like space between the two membranes. Three-dimensional reconstruction of images generated by cryo-electron microscopy indicates that the SpoIIIAG ring has a cup-and-saucer architecture with a 6-nm central pore. Structural modeling of SpoIIIAG generated a 24-member ring with dimensions similar to those of the EM-derived saucer. Point mutations in the predicted oligomeric interface disrupted ring formation in vitro and impaired forespore gene expression and efficient spore formation in vivo. Taken together, our data provide strong support for the model in which the A-Q transenvelope complex contains a conduit that connects the mother cell and forespore. We propose that a set of stacked rings spans the intermembrane space, as has been found for type III secretion systems.

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.

Novel Single Puncture Approach for Simplicity 3 Sacral Plexus Radiofrequency Ablation: Technical Note.

UNLABELLED: Radiofrequency (RF) ablation of the lateral sacral plexus has been used for the treatment of sacroiliac joint pain including as an adjunct to other palliative therapies for the treatment of painful osseous metastasis. The treatment goal is targeted ablation of the dorsal lateral branches of S1-S4. Though several techniques have been described, the Simplicity III (Neurotherm, Middleton, MA) system allows for ablation to be achieved with a single RF probe by utilizing a multi-electrode curved RF probe to create a continuous ablation line across all sacral nerves. In the standard approach, there is sequential introduction of a spinal needle along the desired ablation tract for local anesthesia followed by separate placement of the ablation probe. Though fluoroscopic guidance is utilized, multiple needle passes increase the risk of complication such as bowel perforation or probe insertion through a neural foramen. It may also extend procedure time and increase radiation dose. We illustrate a technique for Simplicity III RF ablation of the dorsal sacral plexus using a modified Seldinger approach for treatment of a patient with sacroiliac joint pain due to osseous renal cell carcinoma metastasis. The desired ablation tract is initially anesthetized via a hollow micropuncture needle. The needle is then exchanged for a peelaway sheath. The RF probe is inserted through the peelaway sheath thus ensuring the probe is placed precisely along the previously anesthetized tract allowing the procedure to be completed using a single percutaneous puncture. We believe that this approach decreases the risks of bowel perforation, patient discomfort as a result of multiple percutaneous punctures, and procedure time. KEY WORDS: Simplicity 3, sacral plexus ablation, image-guided approach, modified Seldinger, chronic sacral pain, thin wall introducer needle.