Biology subjects

Barrows, J. M.

Publications and source records attributed to Barrows, J. M..

4 recordsLinked to original sources

bioRxiv2024

The Cell Division Protein FzlA Performs a Conserved Function in Diverse Alphaproteobacteria

In almost all bacteria, the tubulin-like GTPase FtsZ polymerizes to form a "Z-ring" that marks the site of division. FtsZ recruits other proteins, collectively known as the divisome, that together remodel and constrict the envelope. Constriction is driven by peptidoglycan (PG) cell wall synthesis by the glycosyltransferase FtsW and the transpeptidase FtsI (FtsWI), but these enzymes require activation to function. How recruitment of FtsZ to the division site leads to FtsWI activation and constriction remains largely unknown. Previous work in our laboratory demonstrated that an FtsZ-binding protein, FzlA, is essential for activation of FtsWI in the alphaproteobacterium Caulobacter crescentus. Additionally, we found that FzlA also binds to a DNA translocase called FtsK, suggesting that it may link constriction activation to chromosome segregation. FzlA is conserved throughout alphaproteobacteria but has only been examined in detail in C. crescentus. Here, we explored whether FzlA function is conserved in diverse alphaproteobacteria. We assessed FzlA homologs from Rickettsia parkeri and Agrobacterium tumefaciens, and found that, similar to C. crescentus FzlA, they bind directly to FtsZ and localize to midcell. The FtsZ-FzlA interaction interface is conserved, as we demonstrated that FzlA from each of the three species examined can bind to FtsZ from any of the three in vitro. Additionally, we determined that A. tumefaciens FzlA can fulfill the essential function of FzlA when produced in C. crescentus, indicating conservation of function. These results suggest that FzlA serves as an important regulator that coordinates chromosome segregation with envelope constriction across diverse alphaproteobacteria. ImportanceCell division is essential for bacterial replication and must be highly regulated to ensure robust remodeling of the cell wall in coordination with segregation of the genome to daughter cells. In Caulobacter crescentus, FzlA plays a major role in regulating this process by activating cell wall synthesis in a manner that couples constriction to chromosome segregation. FzlA is broadly conserved in alphaproteobacteria suggesting it plays a similar function across this class of bacteria. Here we have shown that, indeed, FzlA biochemical interactions and function are conserved in diverse alphaproteobacteria. Because FzlA is conserved in alphaproteobacterial human pathogens, understanding this protein and its interactome could present therapeutic benefits by identifying potential antibiotic targets to treat infections.

microbiology↗

bioRxiv2023

Intrinsic and extrinsic factors regulate FtsZ function in Caulobacter crescentus

Bacterial cell division is crucial for replication and requires careful coordination via a complex set of proteins collectively known as the divisome. The tubulin-like GTPase FtsZ is the master regulator of this process and serves to recruit downstream divisome proteins and regulate their activities. Upon arrival at mid-cell, FtsZ associates with the membrane via anchoring proteins and exhibits treadmilling motion, driven by its GTP binding and hydrolysis activities. Treadmilling is proposed to play a role in Z-ring condensation, as well as in distribution and regulation of peptidoglycan (PG) cell wall remodeling enzymes. FtsZ polymer superstructure and dynamics are central to its function, yet their regulation is incompletely understood. We sought to address these gaps in knowledge by modulating intrinsic and extrinsic regulators of FtsZ and evaluating their effects in vitro and in cells, alone and in combination. To do this, we leveraged the cell cycle control features of Caulobacter crescentus. We observed that Caulobacter FtsZ variants that abrogate GTP hydrolysis impact FtsZ dynamics and Z-ring positioning, with little to no effect on Z-ring structure or constriction. Production of an FtsZ variant lacking its disordered C-terminal linker ({Delta}CTL) resulted in aberrant Z-ring dynamics and morphology, misregulated PG metabolism, and cell lysis. Combining {Delta}CTL and GTPase mutations was additive, suggesting they each act independently to control the Z-ring. Modulating levels of FtsA resulted in formation of multiple Z-rings that failed to constrict, suggesting roles in regulating both FtsZ superstructure and the activity of downstream divisome components. Collectively, our results indicate that GTP hydrolysis serves primarily to position the Z-ring at mid-cell, the CTL regulates both Z-ring structure and downstream signaling, and FtsA contributes to all aspects of FtsZ assembly and function. The additive effects of these elements are required to support robust and efficient cell division.

microbiology↗

bioRxiv2019

Determinants of FtsZ C-terminal linker-dependent regulation of cell wall metabolism in Caulobacter crescentus

Bacterial cell division requires assembly of a multi-protein machinery or \"divisome\" that remodels the cell envelope to cause constriction. The cytoskeletal protein FtsZ forms a ring-like scaffold for the divisome at the incipient division site. FtsZ has three major regions - a conserved, polymerizing GTPase domain; a C-terminal conserved (CTC) peptide required for binding membrane-anchoring proteins; and a C-terminal linker (CTL) of poor length and sequence conservation. We previously demonstrated that, in Caulobacter crescentus, the CTL regulates FtsZ polymerization in vitro and cell wall metabolism in vivo. To understand the mechanism of CTL-dependent regulation of cell wall metabolism, here we investigated the impact of the CTL on Z-ring structure in cells and employed genetics to identify molecular determinants of the dominant lethal effects of {Delta}CTL. Deleting the CTL specifically resulted in formation of dense, asymmetric, non-ring FtsZ assemblies in vivo. Moreover, we observed that production of an FtsZ variant with the GTPase domain of Escherichia coli FtsZ fused to the CTC of C. crescentus FtsZ phenocopied the effects of C. crescentus {Delta}CTL, suggesting the CTC mediates signaling to cell wall metabolism. Finally, whereas overproduction of ZapA, FzlC, or FtsEX had slight protective effects against {Delta}CTL, depletion of FtsA partially suppressed the effects of {Delta}CTL. From these results, we propose that the cell wall misregulation downstream of {Delta}CTL results from its aberrant assembly properties and is propagated through the interaction between the CTC of FtsZ and FtsA. Our study provides mechanistic insights into CTL-dependent regulation of cell wall enzymes downstream of FtsZ.\n\nImportanceBacterial cell division is essential and requires the recruitment and regulation of a complex network of proteins needed to initiate and guide constriction and cytokinesis. FtsZ serves as a master regulator for this process, and its function is highly dependent on both its self-assembly into a canonical \"Z-ring\" and interaction with protein binding partners, which results in the activation of enzymes that remodel the cell wall to drive constriction. Using mutants of FtsZ and its binding partners, we have established the role of its C-terminal linker domain in regulating Z-ring organization, as well as the requirement for its C-terminal conserved peptide and interaction with the membrane-anchoring protein FtsA for regulating cell wall remodeling for constriction.

microbiology↗

bioRxiv2019

Glycolysis upregulation is neuroprotective as a compensatory mechanism in ALS

Amyotrophic Lateral Sclerosis (ALS), is a fatal neurodegenerative disorder, with TDP-43 inclusions as a major pathological hallmark. Using a Drosophila model of TDP-43 proteinopathy we found significant alterations in glucose metabolism including increased pyruvate, suggesting that modulating glycolysis may be neuroprotective. Indeed, a high sugar diet improves locomotor and lifespan defects caused by TDP-43 proteinopathy in motor neurons or glia, but not muscle, suggesting that metabolic dysregulation occurs in the nervous system. Overexpressing human glucose transporter GLUT-3 in motor neurons mitigates TDP-43 dependent defects in synaptic vesicle recycling and improves locomotion. Furthermore, PFK mRNA, a key indicator of glycolysis, is upregulated in flies and patient derived iPSC motor neurons with TDP-43 pathology. Surprisingly, PFK overexpression rescues TDP-43 induced locomotor deficits. These findings from multiple ALS models show that mechanistically, glycolysis is upregulated in degenerating motor neurons as a compensatory mechanism and suggest that increased glucose availability is protective.

neuroscience↗