Journal: Advanced Science

Article Title: Deterministic Single Cell Encapsulation in Asymmetric Microenvironments to Direct Cell Polarity

doi: 10.1002/advs.202206014

Figure Lengend Snippet: Asymmetric cell‐matrix adhesion leads to polarized membrane tension in single cells. A) Measurement of membrane tension at 1 day after encapsulation of MSCs in compartmentalized microgels. Fluorescence lifetime imaging microscopy (FLIM) was used to evaluate the decay lifetime ( τ ) of the lipid tension reporter on the cell membrane (Figure , Supporting Information). i) Representative image showing τ across different regions (angles in counterclockwise directions from 0 0 ) of the cell membrane in a microgel consisting of ≈60 µM 1% w/v alginate‐RGD (RGD 1 ) on the left side and unmodified RGD (RGD 0 ) on the right side. A small fraction of alginate‐rhodamine was added to RGD 1 to distinguish it from the RGD 0 compartment. Scale bar = 5 µm. ii) τ values from MSCs in gels. n = 10 cells. ** p < 0.01 via Welch's ANOVA, followed by Dunnett's T3 multiple comparison test. Data are shown as mean ± s.d. B) Quantification of F‐actin in F‐tractin‐tdTomato + MSCs. (i) Representative images showing F‐tractin tdTomato (green) signals in gel‐coated MSCs. Both 2D maximum‐projected and 3D reconstructed images are shown. The dotted line indicates the boundary between the two compartments. Scale bar = 5 µm. ii) Ratio of F‐tractin‐tdTomato mean intensity between left (L) and right (R) compartments. iii) Ratio of tdTomato total intensity between the two compartments. n = 18 cells. *** p < 0.001 via Welch's T‐test. C) Quantification of the ratio between the semi‐major axis (a x , x: left or right) and the semi‐minor axis (b) of each cell in between two compartments. Each image was taken by reconstructing a confocal z‐stack, orienting the stack so that the boundary plane between the two compartments is vertical, and taking the midplane normal to the boundary plane. (Top) Representative images, scale bar = 5 µm. (Bottom) Quantification from n = 15 cells per group. ns: not significant, * p < 0.05, **** p < 0.0001 via Welch's ANOVA, followed by Dunnett's T3 multiple comparison test. D) Finite element analysis showing the distribution of tension ( σ ) within the gel when a cell undergoes non‐uniform isotropic volume expansion with 2.7 µm displacement towards RGD 1 (left) and 1.6 µm towards RGD 0 (right).

Article Snippet: Cells in gels were incubated with 1 µM of Flipper‐TR lipid membrane tension probe (Cytoskeleton, Inc.) [ ] in complete DMEM for 30 min. One of the gels was labeled with red (rhodamine) fluorescence to distinguish between two compartments.

Techniques: Fluorescence, Imaging, Microscopy

Journal: Advanced Science

Article Title: Deterministic Single Cell Encapsulation in Asymmetric Microenvironments to Direct Cell Polarity

doi: 10.1002/advs.202206014

Figure Lengend Snippet: Cdc42 mediates the ability of single MSCs to respond to the asymmetric presentation of integrin ligands. The following inhibitors against small GTPases were tested in gel‐coated MSCs with asymmetric RGD presentation after 1 day in culture in the basal medium: ML141 (10 µM; Cdc42 inhibitor), NSC23766 (NSC; 10 µM; Rac1 inhibitor), Rhosin (30 µM; RhoA inhibitor). DMSO (1:1000 dilution) was used as a negative control. A) Cell volume and sphericity analysis after 2 h drug treatment. i) Cytoplasmic volume, ii) nuclear volume, iii) cell sphericity, and iv) nucleus sphericity. n = 3 independent experiments. * p < 0.05, ** p < 0.01 via nested one‐way ANOVA, followed by Tukey's multiple comparison test. B) Membrane tension analysis with the lipid tension reporter by FLIM after 2 h drug treatment. i) Mean decay lifetime ( τ ) values per cell. τ values across different regions (angles in counterclockwise directions from 0 0 ) of the cell membrane after treatment with ii) DMSO and iii) ML141. n = 12 cells. n.s.: not significant, **** p < 0.0001 via Welch's ANOVA, followed by Dunnett's T3 multiple comparison test. C) Gene expression analysis after 10‐day culture in the medium supplemented with mixed osteogenic and adipogenic cocktails. The inhibitors were added throughout the culture. Osteogenic markers i) Alp , ii) Runx2 , and adipogenic marker iii) Pparg1 . n = 3 independent experiments. * p < 0.05, *** p < 0.001 via ordinary one‐way ANOVA, followed by Tukey's multiple comparison test. All data are shown as mean ± SD.

Article Snippet: Cells in gels were incubated with 1 µM of Flipper‐TR lipid membrane tension probe (Cytoskeleton, Inc.) [ ] in complete DMEM for 30 min. One of the gels was labeled with red (rhodamine) fluorescence to distinguish between two compartments.

Techniques: Negative Control, Expressing, Marker

Journal: bioRxiv

Article Title: Lipid packing and local geometry influence septin curvature sensing

doi: 10.1101/2025.02.12.637894

Figure Lengend Snippet: Lipid packing is modulated by lipid composition, both in silico and in vitro. (A) Cartoon representations of four lipid compositions with different degrees of lipid packing. (B) Full lipid compositions for L, M1, M2, and T. Lipid abbreviations : 1,2-dioleoyl-sn- glycero-3-phosphocholine (DOPC); Soy phosphatidylinositol (PI); 1,2-dioleoyl-sn- glycero-3-phospho-(1’-myo-inositol-4’,5’-bisphosphate) (PIP2); cholesterol (CHL); and 1- palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC). (C) . Snapshot of simulated lipid bilayer sheet and an example of a lipid packing defect where headgroups are in blue (DOPC), red (PIP2) and pink (PI). (D) Average lipid packing defect area for four lipid compositions, measured from simulations using open-source software PackMem. N = 3 independent simulations per composition. (E) Density of lipid packing defects measured for 11 x 11 nm membrane sheets of each lipid composition. (F) Schematic for SLB production: small unilamellar vesicles (SUVs) are incubated with silica microspheres of defined size. SUVs assemble onto the surface of microspheres and excess are washed away to produce bilayers. (G) In vitro reconstitution of lipid compositions onto spherical supported lipid bilayers (SLB) with FLIPPER-TR incorporated at 1 mol%. Fluorescence lifetimes measured by FLIM for each lipid composition on 1 µm and 5 µm SLBs. All differences between lipid compositions of the same curvature and between 1 and 5 µm SLBs of the same lipid composition are significant using unpaired t-test **** p<0.0001. Significance labels shown link to . Representative images of 5 µm SLBs, scale bar = 1 µm.

Article Snippet: For experiments testing the relative packing of lipid compositions in vitro, a final concentration of 2 mol% FLIPPER-TR (Spirochrome) dissolved in fresh DMSO was added to lipid mixtures before drying.

Techniques: In Silico, In Vitro, Software, Membrane, Incubation, Fluorescence

Journal: Science Advances

Article Title: External strain on the plasma membrane is relayed to the endoplasmic reticulum by membrane contact sites and alters cellular energetics

doi: 10.1126/sciadv.ads6132

Figure Lengend Snippet: ( A ) Schematic diagram illustrating the experimental approach used to mechanically stimulate monolayers of mouse tendon cells. ( B ) Representative FLIM images of PM tension probed by Flipper-TR in tendon cells with or without cyclic mechanical stimulation. Each bottom panel is an enlargement of the dashed boxed area in the corresponding top panel. ( C ) Distribution of fluorescence lifetime of Flipper-TR after selecting the PM as the region of interest (ROI) between the static group and the stretched group ( n = 25 cells per group, each with at least two ROIs, from three independent experiments). ( D ) Representative FLIM images of overall ER (top) stained with ER Flipper-TR in tendon cells with or without cyclic mechanical stimulation and their enlargements showing ER tubules (middle), as indicated by the red arrows in the top images, and areas of ER sheets (bottom), as indicated by purple dashed rectangles in the top images. ( E ) Distribution of fluorescence lifetime of ER Flipper-TR selecting the overall ER, ER tubules, or ER sheets, as the ROI between the static group and the stretched group ( n = 24 cells per group, each with at least two ROIs, from three independent experiments). ( F ) Representative FLIM images of mitochondria stained with Mito Flipper-TR (top) and lysosome stained with Lyso Flipper-TR (bottom), in tendon cells with or without cyclic mechanical strain. Each top right panel is an enlargement of the red dashed boxed area in the yellow dashed boxed bottom left panel. ( G ) Distribution of fluorescence lifetime of Mito Flipper-TR and Lyso Flipper-TR between static group and stretched group ( n = 30 cells per group, each with at least three ROIs, from three independent experiments). Line marks the mean of the distribution. Scale bars, 1 μm. ** P < 0.01; *** P < 0.001; n.s., not significant by Student’s t test.

Article Snippet: Samples were then incubated with 1 μM Flipper-TR (SC020, Spirochrome), 1 μM ER Flipper-TR (SC021, Spirochrome), 1 μM Mito Flipper-TR (SC023, Spirochrome), or 1 μM Lyso Flipper-TR (SC022, Spirochrome) for 15 min and washed with live-cell imaging solution (A14291DJ, Invitrogen) before imaging.

Techniques: Fluorescence, Staining