Determination of subunit stoichiometry using single molecule microscopy.

<p>A) and B) 20 consecutive images of a streams acquisition of one cell expressing FloT-YFP, 20 ms time intervals. The triangles indicate a relatively stationary focus that bleaches during the stream acquisition. White bar 2 μm. C to E) Graphs showing bleaching events. One individual bleaching step is indicated in panel C), an event of addition of a molecule (positive increase in fluorescence) in panel D), panel E) shows all clear bleaching steps in an acquisition.</p

The soluble part of FloA (FloAsp) forms multimers.

<p>A) SDS-PAGE showing purification by Ni-NTA chromatography of FloAsp carrying a 6xHis tag at the N-terminus, lane 1 flow through, lane 2 wash step, lane 3 peak elution. B) Circular dichroism analysis of FloAsp (25 μM). C) Size-exclusion chromatography (SEC) of FloAsp; elution of marker proteins and of the void peak are indicated by arrows, numbered letters indicate fractions, which are shown in D) SDS PAGE showing corresponding fractions from SEC. E-F) SDS PAGE of fractions from 5 to 20% (w/v) sucrose gradient centrifugation, position of marker proteins is indicated by arrows, size of denatured proteins is indicated by the marker lane. E) Loading of fraction B5 (dimeric FloAsp) from SEC, F) loading of fraction A5 (multimeric FloAsp); the last lane of the SDS PAGE shows the 35 and 25 kDa proteins from the marker lane. G) Electron microscopy (EM, uranyl acetate negative stain) of the LMW fraction of Strep-FloAsp, H) EM of the HMW fraction of Strep-FloAsp, scale bars 100 nm.</p

Localization of flotillins and associated protein in exponentially growing <i>B</i>. <i>subtilis</i> cells expressing two protein fusions.

<p>A) FloA-CFP and KinC-YFP, Nomarski-DIC, overlay of Nomarski-DIC, FloA-CFP (green) and KinC-YFP. B) FloT-CFP, FloA-YFP, overlay of Nomarski-DIC and FloT-CFP (red) and FloA-YFP (green). C) KinC-YFP, FloT-CFP, overlay of Nomarski-DIC and KinC-YFP (red) and FloT-CFP (green). D) NfeD2-YFP and FloT-CFP, overlay of Nomarski-DIC and NfeD2-YFP (red) and FloT-CFP (green). E) SecA-YFP and FloT-CFP, overlay of Nomarski-DIC and SecA-YFP (red) and FloT-CFP (green), F) overlay of FtsH-YFP (green) and FloA-CFP (red), images of the two panels were taken with an interval of 200 ms. White triangles indicate cases of colocalization, white bars 2 μm.</p

Localization of eukaryotic flotillins, imaged by STED microscopy.

<p>A) FloA-GFP expressed in <i>Aspergillus nidulans</i>, B) reggie-1 (flotillin-2)-GFP in HeLa cells, C) Sizes of flotillin assemblies in various organisms, standard deviation is indicated by bars, n = 50 or more per protein, D) reggie-1 (flotillin-2)-GFP in MCF-7 cells.</p

Dynamics of <i>Bacillus</i> flotillin structures.

<p>A) overlay of a bright field image of a single <i>Bacillus</i> cells with the first frame of fluorescence (green signals) and of 9 trajectories, each having a different colour, of YFP tagged protein FloA, displayed over a time course of 8 seconds. B) individual mean squared displacement curves of the 9 particles observed (in corresponding colours) C) Example of about 50 tracks in 2 <i>Bacillus</i> cells, which are analysed in D) MSD curves for FloA; E) MSD curves of FloT for an experiment analogous to that shown in A) and C). F) weighted mean over all tracked MSD curves of FloA (3152 tracks, red curve) and of FloT (N = 1024, black curve). Error bars indicate the confidence of the mean values, (given by the weighted standard deviation, divided by the number of degrees of freedom). Dotted lines show the fits to the diffusion constants. G) Plot like in panel F), but differentiated into movement in the y-direction or in the x-direction for FloA-YFP; H) distribution of instantaneous velocities of FloA and along the perpendicular (v_x, blue bars) and longitudinal (v_y, red bars) cell axis, No = number. I) same as H) for FloT. For FloA, the tails of the distribution are visibly broader than for FloT, indicating a higher variance and therefore a higher diffusion constant of FloA.</p