Nathan W. Goehring

Diverse Paths to Midcell: Assembly of the Bacterial Cell Division Machinery

At the heart of bacterial cell division is a dynamic ring-like structure of polymers of the tubulin homologue FtsZ. This ring forms a scaffold for assembly of at least ten additional proteins at midcell, the majority of which are likely to be involved in remodeling the peptidoglycan cell wall at the division site. Together with FtsZ, these proteins are thought to form a cell division complex, or divisome. In Escherichia coli, the components of the divisome are recruited to midcell according to a strikingly linear hierarchy that predicts a step-wise assembly pathway. However, recent studies have revealed unexpected complexity in the assembly steps, indicating that the apparent linearity does not necessarily reflect a temporal order. The signals used to recruit cell division proteins to midcell are diverse and include regulated self-assembly, protein-protein interactions, and the recognition of specific septal peptidoglycan substrates. There is also evidence for a complex web of interactions among these proteins and at least one distinct subcomplex of cell division proteins has been defined, which is conserved among E. coli, Bacillus subtilis and Streptococcus pneumoniae.

Polarization of PAR proteins by advective triggering of a pattern-forming system.

Patterning of Caenorhabditis elegans zygotes involves passive as well as active mechanisms. In the Caenorhabditis elegans zygote, a conserved network of partitioning-defective (PAR) polarity proteins segregates into an anterior and a posterior domain, facilitated by flows of the cortical actomyosin meshwork. The physical mechanisms by which stable asymmetric PAR distributions arise from transient cortical flows remain unclear. We present evidence that PAR polarity arises from coupling of advective transport by the flowing cell cortex to a multistable PAR reaction-diffusion system. By inducing transient PAR segregation, advection serves as a mechanical trigger for the formation of a PAR pattern within an otherwise stably unpolarized system. We suggest that passive advective transport in an active and flowing material may be a general mechanism for mechanochemical pattern formation in developmental systems.

Premature targeting of cell division proteins to midcell reveals hierarchies of protein interactions involved in divisome assembly

In order to divide, the bacterium Escherichia coli must assemble a set of at least 10 essential proteins at the nascent division site. These proteins localize to midcell according to a linear hierarchy, suggesting that cell division proteins are added to the nascent divisome in strict sequence. We previously described a method, ‘premature targeting’, which allows us to target a protein directly to the division site independently of other cell division proteins normally required for its localization at midcell. By systematically applying this method to probe the recruitment of and associations among late cell division proteins, we show that this linear assembly model is likely incorrect. Rather, we find that the assembly of most of the late proteins can occur independently of ‘upstream’ proteins. Further, most late proteins, when prematurely targeted to midcell, can back‐recruit upstream proteins in the reverse of the predicted pathway. Together these observations indicate that the late proteins, with the notable exception of the last protein in the pathway, FtsN, are associated in a hierarchical set of protein complexes. Based on these observations we present a revised model for assembly of the E. coli division apparatus.

IcsA, a polarly localized autotransporter with an atypical signal peptide, uses the Sec apparatus for secretion, although the Sec apparatus is circumferentially distributed

Asymmetric localization of proteins is essential to many biological functions of bacteria. Shigella IcsA, an outer membrane protein, is localized to the old pole of the bacillus, where it mediates assembly of a polarized actin tail during infection of mammalian cells. Actin tail assembly provides the propulsive force for intracellular movement and intercellular dissemination. Localization of IcsA to the pole is independent of the amino‐terminal signal peptide (Charles, M., Perez, M., Kobil, J.H., and Goldberg, M.B., 2001, Proc Natl Acad Sci USA 98: 9871–9876) suggesting that IcsA targeting occurs in the bacterial cytoplasm and that its secretion across the cytoplasmic membrane occurs only at the pole. Here, we characterize the mechanism by which IcsA is secreted across the cytoplasmic membrane. We present evidence that IcsA requires the SecA ATPase and the SecYEG membrane channel (translocon) for secretion. Our data suggest that YidC is not required for IcsA secretion. Furthermore, we show that polar localization of IcsA is independent of SecA. Finally, we demonstrate that while IcsA requires the SecYEG translocon for secretion, components of this apparatus are uniformly distributed within the membrane. Based on these data, we propose a model for coordinate polar targeting and secretion of IcsA at the bacterial pole.

Rapid β-lactam-induced lysis requires successful assembly of the cell division machinery

β-lactam antibiotics inhibit penicillin binding proteins (PBPs) involved in peptidoglycan synthesis. Although inhibition of peptidoglycan biosynthesis is generally thought to induce cell lysis, the pattern and mechanism of cell lysis can vary substantially. β-lactams that inhibit FtsI, the only division specific PBP, block cell division and result in growth as filaments. These filaments ultimately lyse through a poorly understood mechanism. Here we find that one such β-lactam, cephalexin, can, under certain conditions, lead instead to rapid lysis at nascent division sites through a process that requires the complete and ordered assembly of the divisome, the essential machinery involved in cell division. We propose that this assembly process (in which the localization of cell wall hydrolases depends on properly targeted FtsN, which in turn depends on the presence of FtsI) ensures that the biosynthetic machinery to form new septa is in place before the machinery to degrade septated daughter cells is enabled. β-lactams that target FtsI subvert this mechanism by inhibiting FtsI without perturbing the normal assembly of the cell division machinery and the consequent activation of cell wall hydrolases. One seemingly paradoxical implication of our results is that β-lactam therapy may be improved by promoting active cell division.

PAR proteins diffuse freely across the anterior–posterior boundary in polarized C. elegans embryos

FRAP reveals that a stable PAR boundary requires balancing diffusive flux of PAR proteins between domains with spatial differences in PAR protein membrane affinities.

Membrane Invaginations Reveal Cortical Sites that Pull on Mitotic Spindles in One-Cell C. elegans Embryos

Asymmetric positioning of the mitotic spindle in C. elegans embryos is mediated by force-generating complexes that are anchored at the plasma membrane and that pull on microtubules growing out from the spindle poles. Although asymmetric distribution of the force generators is thought to underlie asymmetric positioning of the spindle, the number and location of the force generators has not been well defined. In particular, it has not been possible to visualize individual force generating events at the cortex. We discovered that perturbation of the acto-myosin cortex leads to the formation of long membrane invaginations that are pulled from the plasma membrane toward the spindle poles. Several lines of evidence show that the invaginations, which also occur in unperturbed embryos though at lower frequency, are pulled by the same force generators responsible for spindle positioning. Thus, the invaginations serve as a tool to localize the sites of force generation at the cortex and allow us to estimate a lower limit on the number of cortical force generators within the cell.

Mutants, Suppressors, and Wrinkled Colonies: Mutant Alleles of the Cell Division Gene ftsQ Point to Functional Domains in FtsQ and a Role for Domain 1C of FtsA in Divisome Assembly

Cell division in Escherichia coli requires the concerted action of at least 10 essential proteins. One of these proteins, FtsQ, is physically associated with multiple essential division proteins, including FtsK, FtsL, FtsB, FtsW, and FtsI. In this work we performed a genetic analysis of the ftsQ gene. Our studies identified C-terminal residues essential for FtsQs interaction with two downstream proteins, FtsL and FtsB. Here we also describe a novel screen for cell division mutants based on a wrinkled-colony morphology, which yielded several new point mutations in ftsQ. Two of these mutations affect localization of FtsQ to midcell and together define a targeting role for FtsQs alpha domain. Further characterization of one localization-defective mutant protein [FtsQ(V92D)] revealed an unexpected role in localization for the first 49 amino acids of FtsQ. Finally, we found a suppressor of FtsQ(V92D) that was due to a point mutation in domain 1C of FtsA, a domain previously implicated in the recruitment of divisome proteins. However, despite reports of a potential interaction between FtsA and FtsQ, suppression by FtsA(I143L) is not mediated via direct contact with FtsQ. Rather, this mutation acts as a general suppressor of division defects, which include deletions of the normally essential genes zipA and ftsK and mutations in FtsQ that affect both localization and recruitment. Together, these results reveal increasingly complex connections within the bacterial divisome.

Role for the nonessential N terminus of FtsN in divisome assembly

FtsN, the last essential protein in the cell division localization hierarchy in Escherichia coli, has several peculiar characteristics, suggesting that it has a unique role in the division process despite the fact that it is conserved in only a subset of bacteria. In addition to suppressing temperature-sensitive mutations in ftsA, ftsK, ftsQ, and ftsI, overexpression of FtsN can compensate for a complete lack of FtsK in the cell. We examined the requirements for this phenomenon. We found that the N-terminal terminal region (cytoplasmic and transmembrane domains) is critical for suppression, while the C-terminal murein-binding domain is dispensable. Our results further suggest that FtsN and FtsK act cooperatively to stabilize the divisome.

FRAP analysis of membrane-associated proteins: lateral diffusion and membrane-cytoplasmic exchange

Obtaining quantitative kinetic parameters from fluorescence recovery after photobleaching (FRAP) experiments generally requires a theoretical analysis of protein mobility and appropriate solutions for FRAP recovery derived for a given geometry. Here we provide a treatment of FRAP recovery for a molecule undergoing a combined process of reversible membrane association and lateral diffusion on the plasma membrane for two commonly used bleach geometries: stripes, and boxes. Such analysis is complicated by the fact that diffusion of a molecule during photobleaching can lead to broadening of the bleach area, resulting in significant deviations of the actual bleach shape from the desired bleach geometry, which creates difficulty in accurately measuring kinetic parameters. Here we overcome the problem of deviations between actual and idealized bleach geometries by parameterizing, more accurately, the initial postbleach state. This allows for reconstruction of an accurate and analytically tractable approximation of the actual fluorescence distribution. Through simulated FRAP experiments, we demonstrate that this method can be used to accurately measure a broad range of combinations of diffusion constants and exchange rates. Use of this method to analyze the plextrin homology domain of PLC-δ1 in Caenorhabditis elegans results in quantitative agreement with prior analysis of this domain in other cells using other methods. Because of the flexibility, relative ease of implementation, and its use of standard, easily obtainable bleach geometries, this method should be broadly applicable to investigation of protein dynamics at the plasma membrane.