SummaryDuring Bacillus subtilis sporulation, an endocytic-like process called engulfment results in one cell being entirely encased in the cytoplasm of another cell. The driving force underlying this process of membrane movement has remained unclear, although components of the machinery have been characterized. Here we provide evidence that synthesis of peptidoglycan, the rigid, strength bearing extracellular polymer of bacteria, is a key part of the missing force-generating mechanism for engulfment. We observed that sites of peptidoglycan synthesis initially coincide with the engulfing membrane and later with the site of engulfment membrane fission. Furthermore, compounds that block muropeptide synthesis or polymerization prevented membrane migration in cells lacking a component of the engulfment machinery (SpoIIQ), and blocked the membrane fission event at the completion of engulfment in all cells. In addition, these compounds inhibited bulge and vesicle formation that occur in spoIID mutant cells unable to initiate engulfment, as did genetic ablation of a protein that polymerizes muropeptides. This is the first report to our knowledge that peptidoglycan synthesis is necessary for membrane movements in bacterial cells and has implications for the mechanism of force generation during cytokinesis.

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. 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.Engulfment involves dynam...

Quantitative single molecule localization microscopy (qSMLM) is a powerful approach to study in situ protein organization. However, uncertainty regarding the photophysical properties of fluorescent reporters can bias the interpretation of detected localizations and subsequent quantification. Furthermore, strategies to efficiently detect endogenous proteins are often constrained by label heterogeneity and reporter size. Here, a new surface assay for molecular isolation (SAMI) was developed for qSMLM and used to characterize photophysical properties of fluorescent proteins and dyes. SAMI-qSMLM afforded robust quantification. To efficiently detect endogenous proteins, we used fluorescent ligands that bind to a specific site on engineered antibody fragments. Both the density and nano-organization of membrane-bound epidermal growth factor receptors (EGFR, HER2, and HER3) were determined by a combination of SAMI, antibody engineering, and pair-correlation analysis. In breast cancer cell lines, we detected distinct differences in receptor density and nano-organization upon treatment with therapeutic agents. This new platform can improve molecular quantification and can be developed to study the local protein environment of intact cells.

Engineered neural stem cells (NSCs) intrinsically migrating to brain tumors offer a promising mechanism for local therapeutic delivery. However, difficulties in quantitative assessments of NSC migration and in estimates of tumor coverage by diffusible therapeutics have impeded development and refinement of NSC‐based therapies. To address this need, we developed techniques by which conventional serial‐sectioned formalin‐fixed paraffin‐embedded (FFPE) brains can be analyzed in their entirety across multiple test animals. We considered a conventional human glioblastoma model: U251 glioma cells orthotopically engrafted in immunodeficient mice receiving intracerebral (i.c.) or intravenous (i.v.) administrations of NSCs expressing a diffusible enzyme to locally catalyze chemotherapeutic formation. NSC migration to tumor sites was dose‐dependent, reaching 50%–60% of total administered NSCs for the i.c route and 1.5% for the i.v. route. Curiously, the most efficient NSC homing was seen with smaller NSC doses, implying existence of rate‐limiting process active during administration and/or migration. Predicted tumor exposure to a diffusing therapeutic (assuming a 50 µm radius of action) could reach greater than 50% of the entire tumor volume for i.c. and 25% for i.v. administration. Within individual sections, coverage of tumor area could be as high as 100% for i.c. and 70% for i.v. routes. Greater estimated therapeutic coverage was observed for larger tumors and for larger tumor regions in individual sections. Overall, we have demonstrated a framework within which investigators may rationally evaluate NSC migration to, and integration into, brain tumors, and therefore enhance understanding of mechanisms that both promote and limit this therapeutic modality. Stem Cells Translational Medicine 2017;6:1522–1532