Journal: Cells

Article Title: Cdc42 Couples T Cell Receptor Endocytosis to GRAF1-Mediated Tubular Invaginations of the Plasma Membrane

doi: 10.3390/cells8111388

Figure Lengend Snippet: Cdc42 and GRAF1 regulate the formation of tubular structures in activated T cells. ( A ) Jurkat T cells expressing GFP-GRAF1 (left) or GFP-GRAF1-BAR-PH (right) were activated on coverslips coated with functional antibodies against CD3ε and CD28 for 10 min and fixed with paraformaldehyde (PFA). Images are maximum intensity projection of confocal z-stacks ( B ) Left: Maximum intensity projection of a Jurkat T cell expressing GFP-GRAF1 and myc-Cdc42-Q61L and activated as in ( A ). Right: the same cell shown in a 3D reconstruction. ( C ) Blinded quantification of the percentage of cells expressing GFP-GRAF1 and mCherry-tagged Cdc42 variants as indicated and having at least one GRAF1-positive tubule. Each data point represents the mean of an individual experiment, 75–136 cells per experiment. Small horizontal lines indicate mean (±SEM), * p < 0.05; *** p < 0.001; unpaired, two-tailed student’s t-test. ( D ) Jurkat T cells expressing GFP-Cdc42-Q61L, activated and fixed as in ( A ) and stained with an antibody against endogenous GRAF1. Arrows indicate GRAF1-positive tubules. Images representative of at least three independent experiments. Scale bar: 5 μm.

Article Snippet: For fast two-channel live cell imaging, the microscope was equipped with an incubation chamber (Pecon, Erbach, Germany) and W-VIEW GEMINI image splitting optics with filters for spectral separation of GFP and mCherry (Hamamatsu, Hamamatsu, Japan).

Techniques: Expressing, Functional Assay, Two Tailed Test, Staining

Journal: Cells

Article Title: Cdc42 Couples T Cell Receptor Endocytosis to GRAF1-Mediated Tubular Invaginations of the Plasma Membrane

doi: 10.3390/cells8111388

Figure Lengend Snippet: GRAF1- and CTxB-positive tubules contain TCRζ but not transferrin. ( A ) Representative time series of Jurkat T cells expressing myc-Cdc42-Q61L, GFP-GRAF1, and TCRζ-mCherry, and activated on coverslips coated with functional antibodies against CD3ε and CD28 and imaged live at 37˚C. ( B , C ) Jurkat T cells expressing myc-Cdc42-Q61L and GFP-GRAF1 ( B ) or TCRζ-mCherry ( C ) were incubated with Alexa546-Tf ( B ) or CTxB-FITC ( C ) for 10 min and imaged live at 37 °C. ( D ) Quantification of the cells as shown in ( A ), ( B ), and ( D ). Percentage of GRAF1- or CTxB-positive tubules that were also positive for TCRζ-mCherry, Alexa546-Tf, or Lat-mCherry in activated (left, anti-CD3ε and anti-CD28) or resting (right, Poly-L-Lysine) cells. On the right, the dotted horizontal line represents the values for TCRζ in activated cells. Data from at least 45 cells in at least 3 independent experiments. Each data point represents a cell. Small horizontal lines indicate mean (±SEM), **** p < 0.0001, unpaired, n.s. not significant, two-tailed student’s t-test. There were 11 outliers removed for Tf, 8 outliers were removed for Lat-mCherry (resting) using the robust regression and outlier removal (ROUT) method (Q = 0.1%). Scale bar: 5 μm.

Article Snippet: For fast two-channel live cell imaging, the microscope was equipped with an incubation chamber (Pecon, Erbach, Germany) and W-VIEW GEMINI image splitting optics with filters for spectral separation of GFP and mCherry (Hamamatsu, Hamamatsu, Japan).

Techniques: Expressing, Functional Assay, Incubation, Two Tailed Test

Journal: Cells

Article Title: Cdc42 Couples T Cell Receptor Endocytosis to GRAF1-Mediated Tubular Invaginations of the Plasma Membrane

doi: 10.3390/cells8111388

Figure Lengend Snippet: ( A ) Schematic of the PAmCherry-based internalization assay; PAmCherry is photoactivated at the plasma membrane using TIRF illumination for 10 sec. Incorporation of TCRζ-PAmCherry is visualized by the increase in red fluorescence intensity in the GFP-GRAF1 positive tubules. ( B ) Representative time series of the dynamics of the TCRζ-PAmCherry signal before, during, and after photoactivation in Jurkat T cells activated on an antibody-coated coverslip in TIRF illumination. ( C ) Representative image of GFP-GRAF1 and TCRζ-mCherry positive tubules stained with the membrane dye CellMask at 4 °C followed by fixation with PFA at 37 °C. Scale bar: 5 μm.

Article Snippet: For fast two-channel live cell imaging, the microscope was equipped with an incubation chamber (Pecon, Erbach, Germany) and W-VIEW GEMINI image splitting optics with filters for spectral separation of GFP and mCherry (Hamamatsu, Hamamatsu, Japan).

Techniques: Fluorescence, Staining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Primary cilia membrane assembly is initiated by Rab11 and transport protein particle II (TRAPPII) complex-dependent trafficking of Rabin8 to the centrosome

doi: 10.1073/pnas.1018823108

Figure Lengend Snippet: Rab8 localizes to developing cilia but departs mature cilia. (A) Rab8 is an early marker of ciliogenesis. GFP-Rab8a localization in cilia was scored in live RPE GFP-Rab8a cells, and Actub-positive cilia were counted in fixed cells (n = 75 cells). (B) Rab8 accumulates in the growing cilia. One hour after serum withdrawal, RPE GFP-Rab8a cells transiently expressing tRFP-Centrin2 for 24 h were imaged every 10 min by SDC microscopy over 6 h with concurrent imaging in both red and green channels. Representative contrast-adjusted images shown are projections of 20 z-sections (step size 0.5 μm). (Bottom Row) 3D surface rendering. (Scale bar: 5 μm.) (Movie S1). (C) Kinetics of ciliogenesis. Keying on the initiation of nascent cilia, GFP-Rab8a levels in 12 developing cilia were measured from projected z-stacks collected as described in B. Regression analysis was performed on raw data (Inset) to determine the time required for GFP fluorescence to reach maximal levels (106 ± 12 min).

Article Snippet: GFP-Rab8a cilia localization was observed live by epifluorescence microscopy or Marianas spinning disk confocal (SDC) microscopy (Intelligent Imaging Innovations) at 40× magnification.

Techniques: Marker, Expressing, Microscopy, Imaging, Fluorescence

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Primary cilia membrane assembly is initiated by Rab11 and transport protein particle II (TRAPPII) complex-dependent trafficking of Rabin8 to the centrosome

doi: 10.1073/pnas.1018823108

Figure Lengend Snippet: Serum withdrawal triggers Rabin8 association with vesicles that accumulate dynamically at the centrosome before ciliary membrane assembly. (A) Rabin8 localizes to dynamic centrosomal vesicles following serum withdrawal. Serum-starved (60 min) RPE GFP-Rabin8 cells were fixed and stained with the centriolar marker γ-tubulin (Movie S2). (Scale bar: 2 μm.) (B) Serum starvation causes transient association of Rabin8 with membrane vesicles and accumulation at the centrosome mediated by its COOH-terminal domain. Shown is quantification of GFP-Rabin8 and GFP-Rabin81–316 (ΔC)-positive vesicles and centrosomal accumulation in RPE cells (n = 50) (Fig. S2 A and B). (C) The Rabin8 COOH-terminal domain is required for ciliation. GFP-Rab8a–positive cilia were counted in cells transiently expressing control (tRFP), tRFP-Rabin8, and tRFP-Rabin81–316 for 72 h; serum was removed for the last 24 h. GFP-positive cilia were counted only in tRFP-positive cells (n = 50; P < 0.001). (D) Time course of RPE cells accumulating GFP-Rabin8 at the centrosome after serum removal. Fluorescence signal (black) has been inverted in D and E for better viewing of structures. (Scale bar: 2 μm.) n, nucleus. (E) Rabin8 centrosomal targeting precedes Rab8a ciliary membrane formation and is reduced after ciliogenesis. RPE GFP-Rabin8 cells transiently expressing tRFP-Rab8a for 24 h were imaged live every 30 min after serum withdrawal. GFP and tRFP images were collected separately. (Scale bar: 2 μm.)

Article Snippet: GFP-Rab8a cilia localization was observed live by epifluorescence microscopy or Marianas spinning disk confocal (SDC) microscopy (Intelligent Imaging Innovations) at 40× magnification.

Techniques: Staining, Marker, Expressing, Fluorescence