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: Scientific Reports

Article Title: Remote-refocusing light-sheet fluorescence microscopy enables 3D imaging of electromechanical coupling of hiPSC-derived and adult cardiomyocytes in co-culture

doi: 10.1038/s41598-023-29419-w

Figure Lengend Snippet: Synchronized contraction between spatially separated adult-CM in day 0, 1, 2 co-culture imaged under widefield transillumination. ( a–c ) Top images show two merged frames: at peak of contraction (cyan), and at baseline (red), taken t = 2.5 s apart for samples on ( a ) day 0, ( b ) day 1 and ( c ) day 2 of co-culture. Orange arrows indicate some of the adult cardiomyocytes on top of the layer of hiPSC CM co-culture. ( a–c ) Bottom row shows zoomed in ROI indicated by the yellow rectangles in the top row, with green arrows pointing out some of the contraction movement between the merged frames (see Supplementary Video for 50 s timelapse). ( d–f ) Images for days 0, 1, and 2, calculated as the standard deviation across the whole acquisition, indicating movement during acquisition. The yellow rectangles indicate the zoomed in ROI shown in the bottom row of ( a–c ). Scalebar: 100 µm.

Article Snippet: For imaging over a larger FOV than achievable with the LSFM system magnification, complementary brightfield transillumination and epi-fluorescence 2D-timelapse of the hiPSC-CM and adult-CM co-culture was carried out on an inverted widefield microscope (IX73, Olympus) equipped with LED illumination (CoolLED pE300) and a B/W CCD digital camera (Hamamatsu C4742-95-12ER).

Techniques: Co-Culture Assay, Standard Deviation

Journal: Scientific Reports

Article Title: Remote-refocusing light-sheet fluorescence microscopy enables 3D imaging of electromechanical coupling of hiPSC-derived and adult cardiomyocytes in co-culture

doi: 10.1038/s41598-023-29419-w

Figure Lengend Snippet: Widefield fluorescence imaging of calcium transients in adult-CM and hiPSC-CM co-culture on day 0 (left), day 1 (middle) and day 2 (right). Single frames at baseline ( a–c ) and peak of first transient ( d–f ), with frames from the same sample displayed on the same intensity scale. ( h–j ) Standard deviation calculated across the whole acquisition. Orange arrows indicate examples of coupled adult-CM. ( k–m ) Fluorescence intensity variation through hiPSC-CM (blue square ROI in middle row) and adult-CM (orange square ROI in middle row) indicating synchronized calcium transients. Scalebar: 100 µm. The corresponding timelapse is shown in Supplementary Video .

Article Snippet: For imaging over a larger FOV than achievable with the LSFM system magnification, complementary brightfield transillumination and epi-fluorescence 2D-timelapse of the hiPSC-CM and adult-CM co-culture was carried out on an inverted widefield microscope (IX73, Olympus) equipped with LED illumination (CoolLED pE300) and a B/W CCD digital camera (Hamamatsu C4742-95-12ER).

Techniques: Fluorescence, Imaging, Co-Culture Assay, Standard Deviation

Journal: Scientific Reports

Article Title: SAS6-like protein in Plasmodium indicates that conoid-associated apical complex proteins persist in invasive stages within the mosquito vector

doi: 10.1038/srep28604

Figure Lengend Snippet: T. gondii expressing endogenous SAS6L with C-terminal fusion to HA-APEX fixed in host vacuoles and stained for HA (green) and either IMC1 (red, a and c ) or centrin (red, b ) ( a ) 3D-SIM super-resolution images with cell apices facing the viewer showing SAS6L rings (arrows, insets) and apices in profile showing SAS6L flush with the apical IMC. ( b ) Widefield images of parasites at different points of the cell cycle indicated by centrosome number and position (stained for centrin): interphase with single centrosome per cell (i); newly divided centrosomes close together (ii, upper vacuole); divided centrosomes migrated further apart (ii, lower vacuole; and iii); cells with nascent apical complexes (iv, arrowheads). ( c ) Widefield images of parasites showing daughter pellicle formation (stained for IMC1): before daughter pellicle formation (i and ii, upper vacuoles); small pellicle cups (i and ii, lower vacuoles; iii, upper vacuole); mid pellicle development (iii, lower vacuole). Scale bars = ( a ) 5 μm and 500 nm (inset); and ( b , c ) 10 μm.

Article Snippet: P. bergehi images were captured as described for live imaging using a 100x oil immersion objective, and T. gondii images were captured using a Nikon Ti-E widefield inverted microscope with a Hamamatsu Orca-Flash4.0 CMOS camera.

Techniques: Expressing, Staining