Quantitative analysis and modeling of katanin function in flagellar length control

© The Author(s), 2014. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Molecular Biology of the Cell 25 (2014): 3686-3698, doi:10.1091/mbc.E14-06-1116.Flagellar length control in Chlamydomonas reinhardtii provides a simple model system in which to investigate the general question of how cells regulate organelle size. Previous work demonstrated that Chlamydomonas cytoplasm contains a pool of flagellar precursor proteins sufficient to assemble a half-length flagellum and that assembly of full-length flagella requires synthesis of additional precursors to augment the preexisting pool. The regulatory systems that control the synthesis and regeneration of this pool are not known, although transcriptional regulation clearly plays a role. We used quantitative analysis of length distributions to identify candidate genes controlling pool regeneration and found that a mutation in the p80 regulatory subunit of katanin, encoded by the PF15 gene in Chlamydomonas, alters flagellar length by changing the kinetics of precursor pool utilization. This finding suggests a model in which flagella compete with cytoplasmic microtubules for a fixed pool of tubulin, with katanin-mediated severing allowing easier access to this pool during flagellar assembly. We tested this model using a stochastic simulation that confirms that cytoplasmic microtubules can compete with flagella for a limited tubulin pool, showing that alteration of cytoplasmic microtubule severing could be sufficient to explain the effect of the pf15 mutations on flagellar length.This work was funded by National Institutes of Health Grant R01 GM097017

PALM and STORM : unlocking live-cell super-resolution

Live-cell fluorescence light microscopy has emerged as an important tool in the study of cellular biology. The development of fluorescent markers in parallel with super-resolution imaging systems has pushed light microscopy into the realm of molecular visualization at the nanometer scale. Resolutions previously only attained with electron microscopes are now within the grasp of light microscopes. However, until recently, live-cell imaging approaches have eluded super-resolution microscopy, hampering it from reaching its full potential for revealing the dynamic interactions in biology occurring at the single molecule level. Here we examine recent advances in the super-resolution imaging of living cells by reviewing recent breakthroughs in single molecule localization microscopy methods such as PALM and STORM to achieve this important goal.http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1097-028

Cell wall damage reveals spatial flexibility in peptidoglycan synthesis and a non-redundant role for RodA in mycobacteria [preprint]

Cell wall peptidoglycan is a heteropolymeric mesh that protects the bacteria from internal turgor and external insults. In many rod-shaped bacteria, peptidoglycan synthesis for normal growth is achieved by two distinct pathways: the Rod complex, comprised of MreB, RodA and a cognate class B PBP, and the class A PBPs. In contrast to laterally-growing bacteria, pole-growing mycobacteria do not encode an MreB homolog and do not require SEDS protein RodA for in vitro growth. However, RodA contributes to survival of Mycobacterium tuberculosis in some infection models, suggesting that the protein could have a stress-dependent role in maintaining cell wall integrity. Under basal conditions, we find here that the subcellular distribution of RodA largely overlaps with that of the aPBP PonA1, and that both RodA and the aPBPs promote polar peptidoglycan assembly. Upon cell wall damage, RodA fortifies M. smegmatis against lysis and, unlike aPBPs, contributes to a shift in peptidoglycan assembly from the poles to the sidewall. Neither RodA nor PonA1 relocalize; instead, the redistribution of nascent cell wall parallels that of peptidoglycan precursor synthase MurG. Our results support a model in which mycobacteria balance polar growth and cell-wide repair via spatial flexibility in precursor synthesis and extracellular insertion. Importance Peptidoglycan synthesis is a highly successful target for antibiotics. The pathway has been extensively studied in model organisms under laboratory-optimized conditions. In natural environments, bacteria are frequently under attack. Moreover the vast majority of bacterial species are unlikely to fit a single paradigm because of differences in growth mode and/or envelope structure. Studying cell wall synthesis under non-optimal conditions and in non-standard species may improve our understanding of pathway function and suggest new inhibition strategies. Mycobacterium smegmatis, a relative of several notorious human and animal pathogens, has an unusual polar growth mode and multi-layered envelope. In this work we challenged M. smegmatis with cell wall-damaging enzymes to characterize the roles of cell wall-building enzymes when the bacterium is under attack

Deletion of a mycobacterial divisome factor collapses single-cell phenotypic heterogeneity

Summary Paragraph While microorganisms are often studied as populations, the behavior of single, individual cells can have profound consequences. For example, tuberculosis, caused by the bacterial pathogen Mycobacterium tuberculosis, requires months of antibiotic therapy even though the bulk of the bacterial population rapidly dies. Shorter courses lead to high rates of relapse because subpopulations of bacilli can survive despite being genetically identical to those that are easily killed 1. In fact, mycobacteria create variability every time a cell divides, producing daughter cells with different sizes and growth rates 2, 3. The mechanism(s) that underlie this high-frequency variation and how variability relates to survival of the population are unknown. Here we show that mycobacteria actively create heterogeneity. Using a fluorescent reporter and a FACS-based transposon screen, we find that deletion of lamA, a gene of previously unknown function, decreases the amount of heterogeneity in the population by decreasing asymmetric polar growth. LamA has no known homologs in other organisms, but is highly conserved across mycobacterial species. We find that LamA is a member of the mycobacterial division complex (“the divisome”). It inhibits growth at nascent new poles, creating asymmetry in polar growth. The kinetics of killing individual cells that lack lamA are more uniform and more rapid with rifampicin and certain drugs that target the cell wall. Our results show that mycobacteria encode a non-conserved protein that controls the pattern of cell growth, resulting in a population that is both heterogeneous and better able to survive antibiotic pressure

Fighting microbial pathogens by integrating host ecosystem interactions and evolution

International audienceSuccessful therapies to combat microbial diseases and cancers require incorporating ecological and evolutionary principles. Drawing upon the fields of ecology and evolutionary biology, we present a systems-based approach in which host and disease-causing factors are considered as part of a complex network of interactions, analogous to studies of "classical" ecosystems. Centering this approach around empirical examples of disease treatment, we present evidence that successful therapies invariably engage multiple interactions with other components of the host ecosystem. Many of these factors interact nonlinearly to yield synergistic benefits and curative outcomes. We argue that these synergies and nonlinear feedbacks must be leveraged to improve the study of pathogenesis in situ and to develop more effective therapies. An eco-evolutionary systems perspective has surprising and important consequences, and we use it to articulate areas of high research priority for improving treatment strategies

Phosphorylation of the Peptidoglycan Synthase PonA1 Governs the Rate of Polar Elongation in Mycobacteria

Cell growth and division are required for the progression of bacterial infections. Most rod-shaped bacteria grow by inserting new cell wall along their mid-section. However, mycobacteria, including the human pathogen Mycobacterium tuberculosis, produce new cell wall material at their poles. How mycobacteria control this different mode of growth is incompletely understood. Here we find that PonA1, a penicillin binding protein (PBP) capable of transglycosylation and transpeptidation of cell wall peptidoglycan (PG), is a major governor of polar growth in mycobacteria. PonA1 is required for growth of Mycobacterium smegmatis and is critical for M. tuberculosis during infection. In both cases, PonA1’s catalytic activities are both required for normal cell length, though loss of transglycosylase activity has a more pronounced effect than transpeptidation. Mutations that alter the amount or the activity of PonA1 result in abnormal formation of cell poles and changes in cell length. Moreover, altered PonA1 activity results in dramatic differences in antibiotic susceptibility, suggesting that a balance between the two enzymatic activities of PonA1 is critical for survival. We also find that phosphorylation of a cytoplasmic region of PonA1 is required for normal activity. Mutations in a critical phosphorylated residue affect transglycosylase activity and result in abnormal rates of cell elongation. Together, our data indicate that PonA1 is a central determinant of polar growth in mycobacteria, and its governance of cell elongation is required for robust cell fitness during both host-induced and antibiotic stress

Maturing Mycobacterium smegmatis peptidoglycan requires non-canonical crosslinks to maintain shape

In most well-studied rod-shaped bacteria, peptidoglycan is primarily crosslinked by penicillin-binding proteins (PBPs). However, in mycobacteria, crosslinks formed by L,D-transpeptidases (LDTs) are highly abundant. To elucidate the role of these unusual crosslinks, we characterized Mycobacterium smegmatis cells lacking all LDTs. We find that crosslinks generate by LDTs are required for rod shape maintenance specifically at sites of aging cell wall, a byproduct of polar elongation. Asymmetric polar growth leads to a non-uniform distribution of these two types of crosslinks in a single cell. Consequently, in the absence of LDT-mediated crosslinks, PBP-catalyzed crosslinks become more important. Because of this, Mycobacterium tuberculosis (Mtb) is more rapidly killed using a combination of drugs capable of PBP- and LDT- inhibition. Thus, knowledge about the spatial and genetic relationship between drug targets can be exploited to more effectively treat this pathogen

Phosphorylation regulates the rate of cell elongation.

<p><b>(A)</b> Phospho-transfer profiling with the kinase domains of the major serine-threonine protein kinases of <i>M</i>. <i>tuberculosis</i> (<i>Mtb</i> Pkn) reveals that PknB efficiently phosphorylates <i>Mtb</i> MBP-PonA1_cyto. <b>(B)</b><i>M</i>. <i>smegmatis</i> cells that express a T50A allele of <i>ponA1</i> (134 cells; approximate p-value = 0.0145 by the Kolmogorov-Smirnov test) are longer than isogenic wildtype cells (219 cells), while cells that express a T50D allele (139 cells; approximate p-value = 0.0082 by the Kolmogorov-Smirnov test) are shorter than isogenic wildtype, suggesting PonA1’s phosphorylation regulates cell elongation or division. <b>(C)</b> Timelapse microscopy revealed that cells that expressed a T50A (127 cells; approximate p-value = 0.0002 by the Kolmogorov-Smirnov test) allele elongated faster than isogenic wildtype cells (174 cells), which was phenocopied by a truncation of the cytoplasmic tail (<i>Δ</i>cyto; 202 cells; approximate p-value < 0.0001 by the Kolmogorov-Smirnov test). These data suggest that PonA1’s phosphorylation negatively regulates cell elongation. <b>(D)</b> Similarly, phosphorylation status of PonA1 affects total cell length in <i>M</i>. <i>tuberculosis</i>. Cells that expressed a T34A allele (211 cells; approximate p-value = 0.0066 by the Kolmogorov-Smirnov test) exhibited an average cell length 5% longer than isogenic wildtype (202 cells), and cells that expressed a T34D allele (207 cells; approximate p-value < 0.0001 by the Kolmogorov-Smirnov test) were 11% shorter than isogenic wildtype. This suggests that PonA1’s unusual phosphorylation negatively regulates cell elongation in <i>M</i>. <i>tuberculosis</i> as in <i>M</i>. <i>smegmatis</i>. <b>(E)</b><i>Msm</i> cells that encode a T50A,TP- allele of PonA1 are defective for normal cell separation. These cells form short chains of cells with multiple septa (white arrows). These data suggest that PonA1’s phosphorylation may regulate PonA1 TG activity, the remaining functional catalytic activity for this allele, and that alterations to PonA1’s TG activity impact the cell’s peptidoglycan and consequent cleavage of that peptidoglycan.</p

PonA1’s periplasmic domains modulate cell shape in mycobacteria.

<p><b>(A)</b> Replacement of wildtype PonA1 with a PonA1 truncation containing the cytoplasmic tail and TG domain (PonA1_1-360) rescues bacterial survival. Cells exhibit shape changes, but survive, suggesting that PonA1’s TG domain is the sufficient periplasmic domain required for bacterial growth. Cell shape may change due to missing protein-protein interactions that occur along the TP domain. Scale bar, 2 μm. <b>(B)</b> PonA1_1-360 cells (266 cells) are shorter than isogenic wildtype (251 cells; approximate p-value < 0.0001 by the Kolmogorov-Smirnov test), which may indicate that the TP domain plays a role in complex formation that influences cell length. These measurements were taken on a different day than <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005010#ppat.1005010.g002" target="_blank">Fig 2C and 2D</a>, hence the slight difference in isogenic wildtype cell length.</p