Golgi Cisternal Unstacking Stimulates COPI Vesicle Budding and Protein Transport

The Golgi apparatus in mammalian cells is composed of flattened cisternae that are densely packed to form stacks. We have used the Golgi stacking protein GRASP65 as a tool to modify the stacking state of Golgi cisternae. We established an assay to measure protein transport to the cell surface in post-mitotic cells in which the Golgi was unstacked. Cells with an unstacked Golgi showed a higher transport rate compared to cells with stacked Golgi membranes. Vesicle budding from unstacked cisternae in vitro was significantly increased compared to stacked membranes. These results suggest that Golgi cisternal stacking can directly regulate vesicle formation and thus the rate of protein transport through the Golgi. The results further suggest that at the onset of mitosis, unstacking of cisternae allows extensive and rapid vesiculation of the Golgi in preparation for its subsequent partitioning

GM1 and GM2 gangliosides: recent developments.

AbstractGM1 and GM2 gangliosides are important components of the cell membrane and play an integral role in cell signaling and metabolism. In this conceptual overview, we discuss recent developments in our understanding of the basic biological functions of GM1 and GM2 and their involvement in several diseases. In addition to a well-established spectrum of disorders known as gangliosidoses, such as Tay-Sachs disease, more and more evidence points at an involvement of GM1 in Alzheimer's and Parkinson's diseases. New emerging methodologies spanning from single-molecule imaging in vivo to simulations in silico have complemented standard studies based on ganglioside extraction

ERK regulates Golgi and centrosome orientation towards the leading edge through GRASP65

Directed cell migration requires the orientation of the Golgi and centrosome toward the leading edge. We show that stimulation of interphase cells with the mitogens epidermal growth factor or lysophosphatidic acid activates the extracellular signal–regulated kinase (ERK), which phosphorylates the Golgi structural protein GRASP65 at serine 277. Expression of a GRASP65 Ser277 to alanine mutant or a GRASP65 1–201 truncation mutant, neither of which can be phosphorylated by ERK, prevents Golgi orientation to the leading edge in a wound assay. We show that phosphorylation of GRASP65 with recombinant ERK leads to the loss of GRASP65 oligomerization and causes Golgi cisternal unstacking. Furthermore, preventing Golgi polarization by expressing mutated GRASP65 inhibits centrosome orientation, which is rescued upon disassembly of the Golgi structure by brefeldin A. We conclude that Golgi remodeling, mediated by phosphorylation of GRASP65 by ERK, is critical for the establishment of cell polarity in migrating cells

Decoupling polarization of the Golgi apparatus and GM1 in the plasma membrane.

Cell polarization is a process of coordinated cellular rearrangements that prepare the cell for migration. GM1 is synthesized in the Golgi apparatus and localized in membrane microdomains that appear at the leading edge of polarized cells, but the mechanism by which GM1 accumulates asymmetrically is unknown. The Golgi apparatus itself becomes oriented toward the leading edge during cell polarization, which is thought to contribute to plasma membrane asymmetry. Using quantitative image analysis techniques, we measure the extent of polarization of the Golgi apparatus and GM1 in the plasma membrane simultaneously in individual cells subject to a wound assay. We find that GM1 polarization starts just 10 min after stimulation with growth factors, while Golgi apparatus polarization takes 30 min. Drugs that block Golgi polarization or function have no effect on GM1 polarization, and, conversely, inhibiting GM1 polarization does not affect Golgi apparatus polarization. Evaluation of Golgi apparatus and GM1 polarization in single cells reveals no correlation between the two events. Our results indicate that Golgi apparatus and GM1 polarization are controlled by distinct intracellular cascades involving the Ras/Raf/MEK/ERK and the PI3K/Akt/mTOR pathways, respectively. Analysis of cell migration and invasion suggest that MEK/ERK activation is crucial for two dimensional migration, while PI3K activation drives three dimensional invasion, and no cumulative effect is observed from blocking both simultaneously. The independent biochemical control of GM1 polarity by PI3K and Golgi apparatus polarity by MEK/ERK may act synergistically to regulate and reinforce directional selection in cell migration

Drug treatments reveal uncoupled pathways to Golgi apparatus and GM1 polarization.

<p>(<b>A</b>) Composite images (red, GM1; green, Golgi; blue, nuclei) from control experiments (none), incubation with LPA for 30 min, and 30 min pretreatment with the drugs U0126, BFA, or Wortmannin before incubation with LPA (scale bar, 10 µm). (<b>B</b>) Comparison of cumulative distributions of ΔY plasma membrane polarization for all treatments. The plasma membrane is polarized in all cases compared to the unstimulated control, (none, n = 326; LPA, n = 323, p = 0.001; U0126, n = 87, p = 0.001; BFA, n = 104, p = 0.001; Wortmannin, n = 144, p = 0.003), but wortmannin incubation significantly inhibits polarization when compared to LPA incubation alone (LPA vs. Wort, p = 0.001). (<b>C</b>) Cumulative distributions of Golgi angles show that Golgi polarization is inhibited by BFA and U0126, but unaffected by wortmannin. None, n = 293; LPA, n = 322, p = 0.001; U0126, n = 87, p = 0.183; BFA, n = 104, p = 0.736; Wortmannin, n = 144, p = 0.001. *** represents p≤0.001 compared to control using the Kolmogorov-Smirnov test.</p

Analysis of GM1 distribution in response to drug treatments.

<p>(<b>A</b>) GM1 gangliosides labeled with cholera toxin-QDot conjugates at a wound edge after exposure to LPA stimulation in the presence of drugs blocking either GM1 polarization or Golgi apparatus polarization (scale bar, 10 µm). (<b>B</b>) Total amount of GM1 labeling for each condition shown as frequency distributions. The GM1 labeling density is the percentage of total cell area covered by fluorescence signal based on thresholded particle analysis. Each point represents the value for a single cell. *** represents p≤0.001 compared to control using Kolmogorov-Smirnoff statistical tests. (<b>C</b>) Clustering behavior analysis. The average size of aggregates per cell in µm² was plotted against GM1 labeling density, and Deming (model II) linear regression was performed to fit the data and determine slope. Slopes and the associated 95% confidence interval were used to determine significance between experimental groups. *** represents p≤0.001 compared to control using Student's t tests. For (<b>B</b>) and (<b>C</b>), none, n = 111; LPA 10 min, n = 151; LPA 30 min, n = 107; U0126, n = 86; BFA, n = 120; Wortmannin, n = 144.</p

Correlation between ΔY and the Golgi angle in individual cells.

<p>The value of ΔY in µm and the absolute value of the Golgi angle are plotted on the <i>y</i> and <i>x</i> axes, respectively. One point corresponds to the value of ΔY and the Golgi angle calculated in a single cell. The Spearman's rank correlation coefficient ρ and associated p value are reported for each condition. None of the conditions has a significant correlation between GM1 polarization and Golgi apparatus polarization, and all slopes are near zero. None, n = 290, <i>ρ</i> = 0.01, p = 0.92; LPA 10 min, n = 150, <i>ρ</i> = −0.06, p = 0.44; LPA 30 min, n = 293, <i>ρ</i> = −0.08, p = 0.16; U0126, n = 87, <i>ρ</i> = 0.11, p = 0.32; BFA, n = 85, <i>ρ</i> = 0.06, p = 0.61; Wortmannin, n = 144, <i>ρ</i> = 0.01, p = 0.95.</p

Quantification of Golgi apparatus and GM1 polarization.

<p>(<b>A</b>) Golgi apparatus polarization was calculated as a vector (shown in red) that connects the center of mass of the nucleus (C_{m nuc}.) to the center of mass of the Golgi (C_{m Golgi}). From this Golgi vector, an angle is calculated with reference to the <i>y</i> axis, defined as 0° and facing the wound edge. When compared to the classical method, angles falling from −60° to +60° are the equivalent of an oriented Golgi falling within a 120° angle facing the wound edge (shaded area). (<b>B</b>) Plasma membrane polarization was determined by comparing the weighted fluorescence distribution of QDs in the plasma membrane with the geometric center of mass of the cell. GM1 gangliosides in the plasma membrane were labeled with cholera toxin subunit B conjugated to QDs, represented here as red circles. The weighted center of mass of GM1 QD fluorescence was calculated (C_{m PM}) along with the geometric center of mass of the cell (C_{m cell}). Subtracting the <i>y</i> component of C_{m cell} from the <i>y</i> component of C_{m PM} gives us ΔY, a measure of the shift in plasma membrane polarization towards the scratch.</p

Golgi apparatus and GM1 polarization in response to LPA.

<p>(<b>A</b>) Composite images (red, GM1; green, Golgi; blue, nuclei) from untreated wound-edge cells (none) and wound-edge cells incubated with LPA for 10 min or 30 min (scale bar, 10 µm). (<b>B</b>) Cumulative distributions of ΔY plasma membrane polarization values. At both 10 min and 30 min stimulation, ΔY values are significantly increased with respect to control. None, n = 121; 10 min, n = 152, p = 0.001; 30 min, n = 225, p = 0.001. (<b>C</b>) Cumulative distributions of the absolute values of Golgi angles. Compared to control, the LPA-stimulated Golgi apparatus is polarized after 30 min but not after 10 min. None, n = 111; 10 min, n = 150, p = 0.945; 30 min, n = 116, p = 0.001. *** represents p≤0.001 compared to control using the Kolmogorov-Smirnov test.</p