Synthetic motility and cell shape defects associated with deletions of flotillin/reggie paralogs in Bacillus subtilis and interplay of these proteins with NfeD proteins

Abstract

Flotillin/reggie proteins are membrane-associated proteins present in all kinds of cells and belong to the family of proteins carrying the SPFH (stomatin, prohibitin, flotillin, and HflK/HflC) domain. In addition to this domain of unknown function, flotillin proteins are characterized by the flotillin domain, which is rich in heptad repeats. Bacterial flotillin orthologs have recently been shown to be part of lipid rafts, like their eukaryotic counterparts, and to be involved in signaling events. Double deletions of floT and the gene encoding the second flotillin-like protein in Bacillus subtilis, floA, show strong synthetic defects in cell morphology, motility, and transformation efficiency. The lack of FloT resulted in a marked defect in motility. Using total internal reflection fluorescence (TIRF) microscopy, we show that both proteins localize in characteristic focal structures within the cell membrane, which move in a highly dynamic and random manner but localize independently of each other. Thus, flotillin paralogs act in a spatially distinct manner. Flotillin domains in both FloA and FloT are essential for focal assemblies and for the proper function of flotillins. Both flotillin genes are situated next to genes encoding NfeD proteins. FloT dramatically affects the localization of NfeD2: FloT apparently recruits NfeD2 into the focal assemblies, documenting a close interaction between flotillins and NfeDs in bacteria. In contrast, the localization of NfeD1b is not affected by FloA, FloT, or NfeD2. FloA does not show a spatial connection with the upstream-encoded NfeD1b (YqeZ). Our work establishes that bacterial flotillin-like proteins have overlapping functions in a variety of membrane-associated processes and that flotillin domain-mediated assembly and NfeD proteins play important roles in setting up the flotillin raft-like structures in vivo.

Fig 1

Effects of deletions of flotillin-encoding genes on cell shape and cell division. (A and B) Chain of growing B. subtilis cells during vegetative growth (membrane stain FM4-64) (A) or in stationary phase (B). (C) ΔfloT cells during exponential growth. (D) ΔfloA cells during exponential growth. (E and F) Cells carrying a floT deletion and a floA truncation, in which the transcription of the yqfB gene is under the control of the xylose promoter, growing in the presence (E) or absence (F) of xylose. Note that there is no visual difference between the shape defects under the two conditions. (G) ΔfloT ΔfloA double-mutant cells during exponential growth. Note that lysed cells appear more transparent than living cells. (H) Membrane stain of exponentially growing ΔfloT ΔfloA double-mutant cells. The arrowheads on the right indicate membrane abnormalities. Bars, 2 μm.

Fig 2

Motility of flotillin mutant cells. Shown are sizes of colonies on solid plates containing 0.3% or 0.5% agar that were grown at 37°C for 16 h or 24 h, respectively.

Fig 3

Localization of FloT by TIRF microscopy. (A and B) Localization of FloT-YFP (fully functional fusion) during exponential growth (A) and in stationary phase (B). In the overlays, FloT-YFP is green, the membrane is red, and DNA is blue. (C) Time lapse images of a cell expressing FloT-YFP. A single accumulation (indicated by the arrowheads) moves laterally within the membrane, while polar accumulations show random dynamics. (D) FloT-YFP foci represent structures that are able to fuse; note that the fused focus indicated by the arrowheads is much brighter than the two foci before fusion. The images were taken every 3 s; selected time intervals are labeled in panel C. (E) FloT-CFP and NfeD2-YFP frequently colocalize. For most FloT-CFP foci (red in overlay), there is a corresponding NfeD2-YFP focus (green in overlay; colocalizing proteins appear yellow in the overlay). (F) FloT-CFP and FloA-YFP both localize as dynamic foci, but colocalization (FloT-CFP, red, and FloA-YFP, green in overlay) is rarely observed. Bars, 2 μm.

Fig 4

Localization (YFP fluorescence) of FloA and full-length and truncated versions of FloT in mutant backgrounds. Note that the circles with bars in the central plane indicate images acquired in epifluorescence rather than in TIRF mode. (A) FloT-YFP. (B) FloT-YFP in the absence of FloA. (C and D) The localization of FloT-YFP is not altered by the absence of the C-terminal heptad repeat-rich domain of unknown function (C), but FloT-YFP is uniformly distributed upon loss of the flotillin domain (D). (E) FloT-YFP in the absence of NfeD1b. (F) FloT-YFP in the absence of NfeD2 (note the higher number of foci). (G and H) Combination of the FloT-flotillin domain truncation with the floA deletion results a severe cell shape maintenance defect (G), which is not observed in the floA deletion background combined with FloT ΔC-terminal domain construct (H). (I) FloT-YFP in the absence of the downstream yuaI gene. (J and K) FloA-YFP localizes in cells that are incubated in rich medium as distinct accumulations at the membrane during exponential growth (septa between cells are indicated by white lines) (J) and in stationary phase (K). (L) The number of FloA-YFP accumulations is significantly higher in cells growing in minimal medium. (M and N) FloA-YFP localization is not dependent on FloT (M) or on NfeD2 (N). (O) The depletion of the downstream-encoded yqfB gene does not affect the localization of FloA-YFP. (P) The deletion of the flotillin domain of FloA leads to uniform distribution of the truncated YFP-fused protein. (Q) Combination of the FloA-YFP truncation with a deletion of floT alters cell shape. (R) FloA-YFP in cells with nfeD1b deleted. If nothing else is indicated, cells were grown in LB medium. Bars, 2 μm.

Fig 5

Immunodetection of FloT-YFP and truncation mutants using α-GFP antiserum. The signals present in all lanes corresponding to sizes of 48 (indicated by the arrowhead) and 33 kDa are cross-reactions. The calculated sizes of the three fusion proteins were as follows: FloT-YFP, 82 kDa; FloT ΔC-YFP (with deletion of the C-terminal domain), 79 kDa; FloT ΔfloT ΔC-YFP (with deletion of flotillin and the C-terminal domain), 50 kDa.

Fig 6

Localization of NfeD2 and NfeD1b. (A) NfeD2-YFP. (B) NfeD2-YFP in cells with floT deleted. (C) NfeD1b-YFP. (D to G) NfeD1b-YFP localization is not affected by the absence of FloT (D), FloA (E), or YqfB (F and G). (F) Exponential growth. (G) Stationary phase. Bars, 2 μm.

Fig 7

Localization of flotillin proteins in S2 cells. (A) FloT-YFP forms extensive patches on the cell membrane upon expression in Schneider S2 cells. (B) FloA-YFP also localizes to all membranes, where it shows a nonuniform distribution. (C and D) NfeD2-YFP (C) and NfeD1b-YFP (D) show homogeneous localization to vesicles and membranes. (E) FloT-YFP and FloA-CFP do not colocalize when coexpressed. Bars, 5 μm (2 μm in panel A).