Display Settings:

Items per page
We are sorry, but NCBI web applications do not support your browser and may not function properly. More information

Results: 6

1.
FIGURE 2:

FIGURE 2:. From: Isoform-specific tethering links the Golgi ribbon to maintain compartmentalization.

GRASP inactivation blocks Golgi ribbon maintenance. (A) Cells expressing the indicated KR constructs were irradiated with 535- to 580-nm light at an intensity of 2 W/cm2 for 30 s. We acquired z-stacks at 1-min intervals for 10 min before and 60 min after inactivation. Maximum value Z-projections are shown for the indicated time points. Scale bar, 5 μm. (B) The total number of discrete Golgi objects at each time point was quantified using the ImageJ Analyze particles function (n = 10, mean ± SEM).

Timothy Jarvela, et al. Mol Biol Cell. 2014 January 1;25(1):133-144.
2.
FIGURE 5:

FIGURE 5:. From: Isoform-specific tethering links the Golgi ribbon to maintain compartmentalization.

Expression of the GM130-45 chimera increases marker colocalization. (A) Average value z-projections of immunofluorescence images of endogenous GPP130 and stably expressed GalNAcT2-GFP in control knockdown cells, control knockdown cells expressing GM130-45, and GM130 knockdown cells rescued with GM130-45. Scale bar, 5 μm. (B) Profile plots of normalized pixel intensity of GPP130 (red) and GalNAcT2-GFP (green) corresponding to the region marked with white lines in A. (C) Colocalization of endogenous GPP130 and GalNAcT2-GFP across experimental conditions as measured by ICQ (n ≥ 15 cells, mean ± SEM; *p = 0.002, **p = 0.016, ***p = 8.2 × 10−7). (D) HeLa cells were transfected with ∆NGM130 or GM130-45. Shown is a representative single z-slice of a three-dimensional structured illumination image showing fluorescence patterns of endogenous GPP130 (red) and golgin-97 (green). Scale bar, 5 μm. (E) Normalized values of pixel intensity for GPP130 (red) and golgin-97 (green) along the region indicated by the white lines in D. (F) Colocalization of endogenous GPP130 and golgin-97 is described by the Pearson's correlation coefficient for cells transfected with ∆N GM130-WT or GM130-45 (n ≥ 15, mean ± SEM; *p = 2.9 × 10−11).

Timothy Jarvela, et al. Mol Biol Cell. 2014 January 1;25(1):133-144.
3.
FIGURE 1:

FIGURE 1:. From: Isoform-specific tethering links the Golgi ribbon to maintain compartmentalization.

Inactivation of GRASP proteins blocks Golgi ribbon formation. (A) Cells stably expressing GalNAcT2-GFP were transfected with KR constructs. After 2 d, GalNAcT2-GFP was redistributed to the ER with 20 μg/ml brefeldin A for 20 min. Cells were then quickly washed and placed in imaging medium and irradiated with 535- to 580-nm light at an intensity of 2 W/cm2 for 30 s. Average value z-projections of GalNAcT2-GFP fluorescence are shown for the indicated times after inactivation. Scale bar, 5 μm. (B) Golgi assembly was quantified by determining the size of the largest GalNAcT2-GFP object at each time point, with the threshold set above the level of GalNAcT2-GFP fluorescence in the ER (n = 10, mean ± SEM). (C) Cells were fixed 90 min after BFA washout and stained for GPP130. Fluorescence intensity across the width of the Golgi objects (white line, inset) was measured and compared for the Golgi markers GPP130 (red) and GalNAcT2-GFP (green). Scale bar, 5 μm.

Timothy Jarvela, et al. Mol Biol Cell. 2014 January 1;25(1):133-144.
4.
FIGURE 6:

FIGURE 6:. From: Isoform-specific tethering links the Golgi ribbon to maintain compartmentalization.

Decreased glycan processing in cells expressing the GM130-45 chimera. (A) Cell surface staining for terminal N-acetyl-d-glucosamine levels was performed using Alexa 647–conjugated GSII-lectin. Cells were transfected with the GM130-45 construct were grown for 84 h and then treated with trypsin and passed onto new coverslips for 12 h before fixation. The Golgi marked by GPP130 and myc-tagged ∆N-GM130 or GM130-45 construct levels are also shown. Maximum value z-projections are shown for each channel. Cell boundaries are outlined to the extent that they were evident from a low level of background staining detected upon overexposure in the anti-myc channel. A small degree of fluorescent signal was present beyond these boundaries and most likely represents lectin binding to cells whose boundaries escaped detection or lectin binding to cell debris. Scale bar, 10 μm. (B) Average staining of GSII-lectin normalized to the mean value of control cells (n > 15 cells, mean ± SEM). Only fluorescence within identified cell boundaries was quantitated. Statistical significance was determined using a single-tailed paired Student's t test (*p = 0.00001).

Timothy Jarvela, et al. Mol Biol Cell. 2014 January 1;25(1):133-144.
5.
FIGURE 4:

FIGURE 4:. From: Isoform-specific tethering links the Golgi ribbon to maintain compartmentalization.

The GM130-45 chimera rescues Golgi ribbon integrity after GM130 knockdown. (A) Immunofluorescence staining for cells stably expressing GalNAcT2-GFP that were transfected with control or GM130-specific siRNA 96 h before fixation. Where indicated, the cells were also transfected with the myc-tagged GM130-45 chimera 12 h before fixation. Scale bar, 5 μm. (B) The amount of GRASP65 fluorescence present on the Golgi was determined using GalNAcT2-GFP as a mask. The percentage of the total cellular pool that is present on the Golgi is shown for each condition (n = 10, mean ± SEM; *p < 0.05; n.s. = p > 0.05). (C) The number of Golgi objects present for each condition determined by counting discrete GalNAcT2-GFP–positive objects (n = 10, mean ± SEM; *p < 0.05; n.s., not significant). (D) Average value z-projections of GRASP55 and GPP130 immunofluorescence images in control and mycGM130g45tail-rescued–expressing cells. Expression of GM130-45 increases colocalization of GRASP55 with cis-Golgi marker GPP130, as shown by profile plots (E) and the intensity correlation quotient (F). (G) Time course of Golgi fluorescence recovery after photobleaching in siGM130 and GM130-45–rescued cells. Blue circles encompass the photobleached region. Scale bar, 5 μm. (H) Fluorescence levels in bleached region measured and plotted vs. time in siCtrl-treated cells and siGM130 cells only and siGM130 cells transfected with GM130-45 or ∆N∆C-GM130 (n = 10 cells, mean ± SEM; *p < 0.05; n.s., not significant).

Timothy Jarvela, et al. Mol Biol Cell. 2014 January 1;25(1):133-144.
6.
FIGURE 3:

FIGURE 3:. From: Isoform-specific tethering links the Golgi ribbon to maintain compartmentalization.

GRASPs are required for integrity of distinct levels of the Golgi stack. (A) A schematic of the experiment in which KR constructs were inactivated, and 1 min after inactivation a small segment of the Golgi was photobleached. If the Golgi ribbon remained intact at the cis-Golgi, GPP130-GFP fluorescence was expected to recover. If the trans-Golgi ribbon remained interconnected, GalT-YFP fluorescence was expected to recover. (B, C) Cells expressing the indicated KR constructs were irradiated with 15.8 mW of a 561-nm laser for 45 μs/pixel repeated six times. GPP130-GFP fluorescence to mark the cis-Golgi (B) or GalT-YFP fluorescence to mark the trans-Golgi (C) was recorded at 10-s intervals for 1 min, and then the outlined segment was bleached with 27.3-mW, 488-nm laser light for 20 μs repeated 60 times. Then fluorescence recovery was recorded for a further 10 min at 10-s intervals. Average value z- projections are shown for the indicated time points. Scale bar, 5 μm. (D, E) GPP130-GFP (D) and GalT-YFP (E) fluorescence was measured in the bleached zone and plotted vs. time (n = 10 cells, mean ± SEM). Results were compared for statistical significance using a two-tailed Students t test (*p < 0.05; n.s., not significant). (F) Trans marker GalT-YFP FRAP after being carried out at the indicated times postinactivation of GRASP65-KR (n > 5, mean ± SEM). (G) Cis marker GPP130-GFP FRAP after being carried out at the indicated times postinactivation of GRASP55-KR (n > 5, mean ± SEM).

Timothy Jarvela, et al. Mol Biol Cell. 2014 January 1;25(1):133-144.

Display Settings:

Items per page

Supplemental Content

Recent activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...
Write to the Help Desk