Journal: Molecular Biology of the Cell

Article Title: Phosphorylation of the small heat shock protein HspB1 regulates cytoskeletal recruitment and cell motility

doi: 10.1091/mbc.E22-02-0057

Figure Lengend Snippet: Cell-based model system for evaluating HspB1 function. (A) HspB1 immunoblot of parental WT and CRISPR/Cas9 HspB1-null cells, followed by “rescue” constructs of WT HspB1, nonphosphorylatable Ser15,86A and phosphomimetic Ser15,86E HspB1s expressed in the HspB1-null cells, with vinculin loading control. (B) Widefield microscopy of immunofluorescent localization of the 3 HspB1 rescue constructs in cells on fibronectin-coated coverslips detects diffuse cytoplasmic distribution of HspB1. F-actin images (phalloidin) of the same cells are shown below. (C) Maximum intensity projections of confocal microscopy images of HspB1 immunolocalization (HspB1, green) and vinculin (magenta) in HspB1-null cells expressing the three rescue constructs of WT HspB1 and phosphomutant S15,86A and S15,86E HspB1s, on 47 µm × 47 µm micropattern islands. Insets show zoom Merge image (lower left boxed corner) cytoskeletal distribution of HspB1 detectable in WT and S15,86E HspB1 but not with S15,86A HspB1. Cytoskeletal distribution of HspB1 observed in 40% of WT HspB1 rescue cell images, 3% of S15,86A HspB1 rescue cell images, and 38% of S15,86E HspB1 rescue cell images. (D) Intensity line profiles from cell exterior toward interior (brackets) of vinculin (dashed magenta line) and HspB1 (solid green line). Scale bar 20 microns.

Article Snippet: A Nikon Ti Eclipse inverted widefield microscope with 10× objective (NA 0.45, PlanApo) and Perfect Focus System was used for DIC imaging (with Kohler illumination) and acquisition with Nikon Elements v4.6 software and Andor NeoZyla camera.

Techniques: Western Blot, CRISPR, Construct, Control, Microscopy, Confocal Microscopy, Expressing

Journal: Molecular Biology of the Cell

Article Title: Phosphorylation of the small heat shock protein HspB1 regulates cytoskeletal recruitment and cell motility

doi: 10.1091/mbc.E22-02-0057

Figure Lengend Snippet: HspB1 affects cell spreading in a phosphodependent manner. (A) immunofluorescence microscopy of cells spread on glass coverslips coated with 10 µg/ml fibronectin. Subcellular distribution of HspB1 (top row, cytoplasmic) and vinculin (bottom row, FA) in WT and HspB1-null cells, and in null cells expressing the WT HspB1 rescue construct. (B) Graph of cell area measurements shows the decreased cell spread in HspB1-null cells is rescued by expressing WT HspB1 rescue construct. (C) immunofluorescence localization of HspB1 (top row) in HspB1-null cells, and in null cells expressing the rescue constructs for WT HspB1 and nonphosphorylatable S15,86A HspB1 and phosphomimetic S15,86E HspB1. Vinculin immunofluorescent localizations in same cells (bottom row). (D) Graph of cell area measurements show increased cell spreading in cells expressing WT and S15,86E HspB1, but no difference between HspB1-null cells and cells expressing S15,86A HspB1. Scale bar of 20 microns for widefield fluorescent images. Graphs are mean with standard deviations and unpaired t tests were used to determine p-values of ** p < 0.01, *** p < 0.001.

Article Snippet: A Nikon Ti Eclipse inverted widefield microscope with 10× objective (NA 0.45, PlanApo) and Perfect Focus System was used for DIC imaging (with Kohler illumination) and acquisition with Nikon Elements v4.6 software and Andor NeoZyla camera.

Techniques: Immunofluorescence, Microscopy, Expressing, Construct

Journal: bioRxiv

Article Title: RecN and RecA orchestrate an ordered DNA supercompaction response following ciprofloxacin exposure in Escherichia coli

doi: 10.1101/2024.11.15.623168

Figure Lengend Snippet: All cells were grown in LB at 37°C and imaged at 2-minute intervals using live-cell imaging. ( A and B ) Cells were immobilized in microfluidic channel slides and imaged using widefield microscopy. ( A ) Representative images of HU-mCherry labelled (green) wild-type cells at 0, 8, and 16 minutes after CIP exposure, showing DNA distribution. Scale bar: 5 µm. ( B ) Analysis of DNA distribution along the cells’ long axis before and after CIP, quantified by measuring the distance between the outer bounds of symmetrical fluorescence peaks at 80% of maximum averaged intensity for each time point. Results are averaged from 34-141 cells from a single representative biological replicate (see Materials and methods for detailed explanation). ( C and D ) Cells were immobilized on agar pads and imaged using spinning disk microscopy. Results shown are from images captured 12, 14, 16, and 18 minutes after CIP exposure, averaged from 40 tracked cells from a single representative biological replicate. ( C ) Average HU-mCherry fluorescence intensity along the cells’ long axis. ( D ) Kymograph heat map of relative HU-mCherry intensity distribution over time. A.u., arbitrary unit.

Article Snippet: To examine the cells’ immediate response to CIP exposure, we employed a microfluidic setup to image cells using a Zeiss Axio Observer Z1 widefield inverted microscope with a 63x oil objective (Zeiss Plan Apochromat 1.4 NA, DIC), a Colibri 7 LED light source, a Hamamatsu ORCA-Flash4.0 V3 digital CMOS camera, as well as a heated incubation chamber and mounting frame, both maintained at 37°C.

Techniques: Live Cell Imaging, Microscopy, Fluorescence

Journal: bioRxiv

Article Title: RecN and RecA orchestrate an ordered DNA supercompaction response following ciprofloxacin exposure in Escherichia coli

doi: 10.1101/2024.11.15.623168

Figure Lengend Snippet: All cells were grown in LB at 37°C and imaged at 2-minute intervals using live-cell imaging. ( A and B ) Cells were immobilized in microfluidic channel slides and imaged using widefield microscopy. ( A ) Representative images of ΔrecN cells at 0, 8, and 16 minutes after CIP exposure, showing HU-mCherry fluorescence (green) to represent DNA distribution. Scale bar: 5 µm. ( B ) Analysis of DNA distribution along the cells’ long axis before and after CIP, quantified by measuring the distance between the outer bounds of symmetrical fluorescence peaks at 80% of maximum averaged intensity for each time point. Results are averaged from 17-61 cells from a single representative biological replicate (see Materials and methods for detailed explanation). ( C and D ) Cells immobilized on agar pads and imaged using spinning disk microscopy. Results are shown from images captured 12, 14, 16, and 18 minutes after CIP exposure, averaged from 40 tracked cells from a single representative biological replicate. ( C ) Average HU-mCherry fluorescence intensity along the cells’ long axis. ( D ) Kymograph heat map of relative HU-mCherry intensity distribution over time. A.u., arbitrary unit.

Article Snippet: To examine the cells’ immediate response to CIP exposure, we employed a microfluidic setup to image cells using a Zeiss Axio Observer Z1 widefield inverted microscope with a 63x oil objective (Zeiss Plan Apochromat 1.4 NA, DIC), a Colibri 7 LED light source, a Hamamatsu ORCA-Flash4.0 V3 digital CMOS camera, as well as a heated incubation chamber and mounting frame, both maintained at 37°C.

Techniques: Live Cell Imaging, Microscopy, Fluorescence