Membrane binding properties of the cytoskeletal protein bactofilin

Reviewer #1 (Public review):

Summary:

The investigators undertook detailed characterization of a previously proposed membrane targeting sequence (MTS), a short N-terminal peptide, of the bactofilin BacA in Caulobacter crescentus. Using light microscopy, single molecule tracking, liposome binding assays, and molecular dynamics simulations, they provide data to suggest that this sequence indeed does function in membrane targeting and further conclude that membrane targeting is required for polymerization. While the membrane association data are reasonably convincing, there are no direct assays to assess polymerization and some assays used lack proper controls as detailed below. Since the MTS isn't required for bactofilin polymerization in other bacterial homologues, showing that membrane binding facilitates polymerization would be a significant advance for the field.

Major concerns

(1) This work claims that the N-termina MTS domain of BacA is required for polymerization, but they do not provide sufficient evidence that the ∆2-8 mutant or any of the other MTS variants actually do not polymerize (or form higher order structures). Bactofilins are known to form filaments, bundles of filaments, and lattice sheets in vitro and bundles of filaments have been observed in cells. Whether puncta or diffuse labeling represents different polymerized states or filaments vs. monomers has not been established. Microscopy shows mis-localization away from the stalk, but resolution is limited. Further experiments using higher resolution microscopy and TEM of purified protein would prove that the MTS is required for polymerization.
(2) Liposome binding data would be strengthened with TEM images to show BacA binding to liposomes. From this experiment, gross polymerization structures of MTS variants could also be characterized.
(3) The use of the BacA F130R mutant throughout the study to probe the effect of polymerization on membrane binding is concerning as there is no evidence showing that this variant cannot polymerize. Looking through the papers the authors referenced, there was no evidence of an identical mutation in BacA that was shown to be depolymerized or any discussion in this study of how the F130R mutation might to analogous to polymerization-deficient variants in other bactofilins mentioned in these references.
(4) Microscopy shows that a BacA variant lacking the native MTS regains the ability to form puncta, albeit mis-localized, in the cell when fused to a heterologous MTS from MreB. While this swap suggests a link between puncta formation and membrane binding the relationship between puncta and polymerization has not been established (see comment 1).
(5) The authors provide no primary data for single molecule tracking. There is no tracking mapped onto microscopy images to show membrane localization or lack of localization in MTS deletion/variants. A known soluble protein (e.g. unfused mVenus) and a known membrane bound protein would serve as valuable controls to interpret the data presented. It also is unclear why the authors chose to report molecular dynamics as mean squared displacement rather than mean squared displacement per unit time, and the number of localizations is not indicated. Extrapolating from the graph in figure 4 D for example, it looks like WT BacA-mVenus would have a mobility of 0.5 (0.02/0.04) micrometers squared per second which is approaching diffusive behavior. Further justification/details of their analysis method is needed. It's also not clear how one should interpret the finding that several of the double point mutants show higher displacement than deleting the entire MTS. These experiments as they stand don't account for any other cause of molecular behavior change and assume that a decrease in movement is synonymous with membrane binding.
(6) The experiments that map the interaction surface between the N-terminal unstructured region of PbpC and a specific part of the BacA bactofilin domain seem distinct from the main focus of the paper and the data somewhat preliminary. While the PbpC side has been probed by orthogonal approaches (mutation with localization in cells and affinity in vitro), the BacA region side has only been suggested by the deuterium exchange experiment and needs some kind of validation.

Reviewer #2 (Public review):

Summary:

The authors of this study investigated the membrane-binding properties of bactofilin A from Caulobacter crescentus, a classic model organism for bacterial cell biology. BacA was the progenitor of a family of cytoskeletal proteins that have been identified as ubiquitous structural components in bacteria, performing a range of cell biological functions. Association with the cell membrane is a common property of the bactofilins studied and is thought to be important for functionality. However, almost all bactofilins lack a transmembrane domain. While membrane association has been attributed to the unstructured N-terminus, experimental evidence had yet to be provided. As a result, the mode of membrane association and the underlying molecular mechanics remained elusive.

Liu at al. analyze the membrane binding properties of BacA in detail and scrutinize molecular interactions using in-vivo, in-vitro and in-silico techniques. They show that few N-terminal amino acids are important for membrane association or proper localization and suggest that membrane association promotes polymerization. Bioinformatic analyses revealed conserved lineage-specific N-terminal motifs indicating a conserved role in protein localization. Using HDX analysis they also identify a potential interaction site with PbpC, a morphogenic cell wall synthase implicated in Caulobacter stalk synthesis. Complementary, they pinpoint the bactofilin-interacting region within the PbpC C-terminus, known to interact with bactofilin. They further show that BacA localization is independent of PbpC.

Strengths

These data significantly advance the understanding of the membrane binding determinants of bactofilins and thus their function at the molecular level. The major strength of the comprehensive study is the combination of complementary in vivo, in vitro and bioinformatic/simulation approaches, the results of which are consistent.

Weaknesses:

The results are limited to protein localization and interaction, as there is no data on phenotypic effects. Therefore, the cell biological significance remains somewhat underrepresented.

Author response:

Reviewer #1:

Summary:

The investigators undertook detailed characterization of a previously proposed membrane targeting sequence (MTS), a short N-terminal peptide, of the bactofilin BacA in Caulobacter crescentus. Using light microscopy, single molecule tracking, liposome binding assays, and molecular dynamics simulations, they provide data to suggest that this sequence indeed does function in membrane targeting and further conclude that membrane targeting is required for polymerization. While the membrane association data are reasonably convincing, there are no direct assays to assess polymerization and some assays used lack proper controls as detailed below. Since the MTS isn't required for bactofilin polymerization in other bacterial homologues, showing that membrane binding facilitates polymerization would be a significant advance for the field

We thanks Reviewer #1 for the constructive criticism and will address the points detailed below in a revised version of the manuscript.

Major concerns

(1) This work claims that the N-termina MTS domain of BacA is required for polymerization, but they do not provide sufficient evidence that the ∆2-8 mutant or any of the other MTS variants actually do not polymerize (or form higher order structures). Bactofilins are known to form filaments, bundles of filaments, and lattice sheets in vitro and bundles of filaments have been observed in cells. Whether puncta or diffuse labeling represents different polymerized states or filaments vs. monomers has not been established. Microscopy shows mis-localization away from the stalk, but resolution is limited. Further experiments using higher resolution microscopy and TEM of purified protein would prove that the MTS is required for polymerization.

We do not propose that the MTS is directly involved in the polymerization process, and preliminary transmission electron microscopy (TEM) data show that variants lacking the MTS or carrying amino acid exchanges in the MTS still form polymers when highly overproduced in E. coli and then purified from cell lysates by affinity chromatography. This finding is consistent with the results of previous studies and in line with the finding that bactofilin polymerization is exclusively mediated by the conserved bactofilin domain (Deng et al, Nat Microbiol, 2019). However, under native expression conditions, bactofilin levels are often relatively low, with only a few hundred molecules of BacA measured per cell in C. crescentus (Kühn et al, EMBO J, 2006). Our data indicate that, under this condition, the concentration of BacA on the 2D surface of the cytoplasmic membrane and, potentially, steric contraints induced by membrane curvature, may be required to facilitate its efficient assembly into functional polymeric complexes. We will provide TEM images of purified proteins in a revised version of our manuscript and explain this model in more detail in the Discussion.

In the case of polymer-forming proteins, defined localized signals are typically interpreted as polymeric complexes. An even distribution of the fluorescence signals, by contrast, indicates that the proteins form monomers or, at most, small oligomers that diffuse rapidly within the cell and are thus no longer detected as a stationary focus by widefield microscopy. Our single-molecule data also indicate that proteins that are no longer able to interact with the membrane (as verified by cell fractionation studies and in vitro liposome binding assays) show a high diffusion rate, similar to that measured for the non-polymerizing and non-membrane-bound F130R variant. These results indicate that a loss of membrane binding strongly reduces the ability of BacA to form polymeric assemblies. To support this hypothesis, we will perform additional single-molecule tracking analyses of a freely diffusible and membrane-bound monomeric fluorescent proteins for comparison.

(2) Liposome binding data would be strengthened with TEM images to show BacA binding to liposomes. From this experiment, gross polymerization structures of MTS variants could also be characterized.

We do not have the possibility to perform cryo-electron microscopy studies of liposomes bound to BacA. However, the results of the cell fractionation and liposome sedimentation assays clearly support a critical role of the MTS in membrane binding.

(3) The use of the BacA F130R mutant throughout the study to probe the effect of polymerization on membrane binding is concerning as there is no evidence showing that this variant cannot polymerize. Looking through the papers the authors referenced, there was no evidence of an identical mutation in BacA that was shown to be depolymerized or any discussion in this study of how the F130R mutation might to analogous to polymerization-deficient variants in other bactofilins mentioned in these references.

Residue F130 in the C-terminal polymerization interface of BacA is highly conserved among bactofilin homologs, although its absolute position in the protein sequence may vary, depending on the length of the N-terminal unstructured tail. The papers cited in our manuscript show that an exchange of this conserved phenylalanine residue abolishes polymer formation. We will make this fact clearer in the revised version of the manuscript. Moreover, we will provide gel filtration and transmission electron microscopy data showing that the BacA-F130R variant no longer forms polymers.

(4) Microscopy shows that a BacA variant lacking the native MTS regains the ability to form puncta, albeit mis-localized, in the cell when fused to a heterologous MTS from MreB. While this swap suggests a link between puncta formation and membrane binding the relationship between puncta and polymerization has not been established (see comment 1).

We show that a BacA variant lacking the MTS regains the ability to form membrane-associated foci when fused to the MTS of MreB. In contrast, a similar variant that additionally carries the F130R exchange (preventing its polymerization) shows a diffuse cytoplasmic localization. In addition, we show that the F130R exchange leads to a loss of membrane binding and to a considerable increase in the mobility of the variants carrying the MreB MTS. Together, these results strongly support the hypothesis that membrane binding and polymerization act synergistically to establish localized bactofilin assemblies.

(5) The authors provide no primary data for single molecule tracking. There is no tracking mapped onto microscopy images to show membrane localization or lack of localization in MTS deletion/ variants. A known soluble protein (e.g. unfused mVenus) and a known membrane bound protein would serve as valuable controls to interpret the data presented. It also is unclear why the authors chose to report molecular dynamics as mean squared displacement rather than mean squared displacement per unit time, and the number of localizations is not indicated. Extrapolating from the graph in figure 4 D for example, it looks like WT BacA-mVenus would have a mobility of 0.5 (0.02/0.04) micrometers squared per second which is approaching diffusive behavior. Further justification/details of their analysis method is needed. It's also not clear how one should interpret the finding that several of the double point mutants show higher displacement than deleting the entire MTS. These experiments as they stand don't account for any other cause of molecular behavior change and assume that a decrease in movement is synonymous with membrane binding.

We agree that a more in-depth analysis of the single-molecule-tracking data would be helpful to support our conclusions. We will map the reads on the cells, although the loss of membrane localization of BacA variants with a defective MTS is already obvious in the widefield fluorescence images. Moreover, we will perform additional measurements on soluble mVenus and a membrane-associated variant of mVenus for comparison and address the other issues raised here.

The single-molecule tracking data alone are certainly not sufficient to draw firm conclusions on the relationship between membrane binding and protein mobility. However, our other in vivo and in vitro analyses indicate a very clear correlation of between the mobility of BacA and its ability to interact with the membrane and polymerize (processes that synergistically promote each other).

(6) The experiments that map the interaction surface between the N-terminal unstructured region of PbpC and a specific part of the BacA bactofilin domain seem distinct from the main focus of the paper and the data somewhat preliminary. While the PbpC side has been probed by orthogonal approaches (mutation with localization in cells and affinity in vitro), the BacA region side has only been suggested by the deuterium exchange experiment and needs some kind of validation

The results of the HDX analysis per se are not preliminary and clearly indicate a change in the accessibily of surface-exposed residues in the central bactofilin domain. However, we agree that additional experiments would be required to verify the binding site suggested by these data. However, this aspect is indeed not the main focus of the paper. We included the analysis of the interaction between PbpC and BacA, because we see effects of membrane binding/polymerization on the BacA-PbpC interaction and thus on the physiological function of BacA in C. crescentus.

Reviewer #2:

Summary:

The authors of this study investigated the membrane-binding properties of bactofilin A from Caulobacter crescentus, a classic model organism for bacterial cell biology. BacA was the progenitor of a family of cytoskeletal proteins that have been identified as ubiquitous structural components in bacteria, performing a range of cell biological functions. Association with the cell membrane is a common property of the bactofilins studied and is thought to be important for functionality. However, almost all bactofilins lack a transmembrane domain. While membrane association has been attributed to the unstructured N-terminus, experimental evidence had yet to be provided. As a result, the mode of membrane association and the underlying molecular mechanics remained elusive.

Liu at al. analyze the membrane binding properties of BacA in detail and scrutinize molecular interactions using in-vivo, in-vitro and in-silico techniques. They show that few N-terminal amino acids are important for membrane association or proper localization and suggest that membrane association promotes polymerization. Bioinformatic analyses revealed conserved lineage-specific N-terminal motifs indicating a conserved role in protein localization. Using HDX analysis they also identify a potential interaction site with PbpC, a morphogenic cell wall synthase implicated in Caulobacter stalk synthesis. Complementary, they pinpoint the bactofilin-interacting region within the PbpC C-terminus, known to interact with bactofilin. They further show that BacA localization is independent of PbpC.

Strengths

These data significantly advance the understanding of the membrane binding determinants of bactofilins and thus their function at the molecular level. The major strength of the comprehensive study is the combination of complementary in vivo, in vitro and bioinformatic/simulation approaches, the results of which are consistent.

We thank Reviewer #2 for the positive evaluation of our paper and for the constructive criticism sent to us in the the non-public review. We will address the points raised in a revised version of the manuscript.

Weaknesses:

The results are limited to protein localization and interaction, as there is no data on phenotypic effects. Therefore, the cell biological significance remains somewhat underrepresented.

We agree that it would be interesting to investigate the phenotypic effects caused by a defect of BacA in membrane binding. We will investigate PbpC localization and stalk length in phosphate-limited medium for mutants producing MTS-deficient BacA variants and include these data in the revised version of the manuscript. However, we would like to point out that the relevance of our findings goes beyond the C. cres­centus system, because the MTS and its role for bactofilin function is likely to be conserved in many other species.