Both tails and the centromere targeting domain of CENP-A are required for centromere establishment

The centromere—defined by the presence of nucleosomes containing the histone H3 variant, CENP-A—is the chromosomal locus required for the accurate segregation of chromosomes during cell division. Although the sequence determinants of human CENP-A required to maintain a centromere were reported, those that are required for early steps in establishing a new centromere are unknown. In this paper, we used gain-of-function histone H3 chimeras containing various regions unique to CENP-A to investigate early events in centromere establishment. We targeted histone H3 chimeras to chromosomally integrated Lac operator sequences by fusing each of the chimeras to the Lac repressor. Using this approach, we found surprising contributions from a small portion of the N-terminal tail and the CENP-A targeting domain in the initial recruitment of two essential constitutive centromere proteins, CENP-C and CENP-T. Our results indicate that the regions of CENP-A required for early events in centromere establishment differ from those that are required for maintaining centromere identity

Sites of glucose transporter-4 vesicle fusion with the plasma membrane correlate spatially with microtubules.

In adipocytes, vesicles containing glucose transporter-4 (GLUT4) redistribute from intracellular stores to the cell periphery in response to insulin stimulation. Vesicles then fuse with the plasma membrane, facilitating glucose transport into the cell. To gain insight into the details of microtubule involvement, we examined the spatial organization and dynamics of microtubules in relation to GLUT4 vesicle trafficking in living 3T3-L1 adipocytes using total internal reflection fluorescence (TIRF) microscopy. Insulin stimulated an increase in microtubule density and curvature within the TIRF-illuminated region of the cell. The high degree of curvature and abrupt displacements of microtubules indicate that substantial forces act on microtubules. The time course of the microtubule density increase precedes that of the increase in intensity of fluorescently-tagged GLUT4 in this same region of the cell. In addition, portions of the microtubules are highly curved and are pulled closer to the cell cortex, as confirmed by Parallax microscopy. Microtubule disruption delayed and modestly reduced GLUT4 accumulation at the plasma membrane. Quantitative analysis revealed that fusions of GLUT4-containing vesicles with the plasma membrane, detected using insulin-regulated aminopeptidase with a pH-sensitive GFP tag (pHluorin), preferentially occur near microtubules. Interestingly, long-distance vesicle movement along microtubules visible at the cell surface prior to fusion does not appear to account for this proximity. We conclude that microtubules may be important in providing spatial information for GLUT4 vesicle fusion

Movement of vesicles long distances prior to fusion is observed infrequently.

<p>Adipocyte transfected with mCherry-IRAP-pHluorin was serum-starved prior to stimulation with 100 nM insulin. Images were acquired using TIRF microscopy at an acquisition rate of 20 frames per 1 s, and a kymograph was generated. mCherry (magenta) allows visualization of the vesicle prior to fusion with the plasma membrane, at which time the pHluorin (green) intensity increases. The overlay of magenta and green appears white. The vesicle on the right represents an example of a rare vesicle fusion event that is preceded by a long-distance movement. On the left, a second vesicle approaches the surface in the same vicinity as the first but fuses without detected lateral movement.</p

Sites of IRAP-pHluorin fusion are spatially correlated with microtubules present in the TIRF illumination zone.

<p>Adipocytes co-transfected with IRAP-pHluorin and mCherry-tubulin were serum-starved prior to stimulation with 100 nM insulin. Histogram of distance of fusion events from a microtubule obtained using TIRF microscopy and a Dual-view insert at an acquisition rate of 20 frames per 1 s (red, fusions; blue, random locations). Center of bin (width = 0.2 µm) is indicated. Error bars represent the standard deviation resulting from bootstrapping (n = 100 repetitions) the fusion data.</p

TIRF microscopy reveals a population of highly curved microtubules at the surface of 3T3-L1 adipocytes.

<p>Adipocyte transfected with mCherry-tubulin was serum-starved prior to stimulation with 100 nM insulin at t = 0 min. Images were acquired using TIRF microscopy at 1 frame per 10 s. Elapsed time from insulin addition is indicated. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043662#pone.0043662.s007" target="_blank">Video S2</a>. (A) (Upper) Unprocessed and (Lower) background-subtracted, inverted contrast images of an adipocyte at the indicated times following insulin stimulation. Microtubule contours are overlaid in blue. Scale bar is 10 µm. (B) Contours for microtubules at least 3 µm in length were replotted to share the same origin (0 min, 5 contours; 5 min, 8 contours; 10 min, 19 contours; 15 min, 15 contours). (C) Cosine correlation function for microtubule contours shown in (B), see supplement for details (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043662#pone.0043662.s016" target="_blank">Materials S1</a>: Cosine correlation function, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043662#pone.0043662.s003" target="_blank">Figure S3</a>). (D) The average CCF up to the minimum contour length for microtubule contours shown in (C) are plotted at 0 (red), 5 (orange), 10 (green), and 15 (blue) minutes post-insulin. Error bars represent the 95% confidence interval.</p

Curved regions of microtubules are actively displaced.

<p>Adipocyte was transfected with 3×GFP-EMTB in order to visualize microtubules. Images were acquired using TIRF microscopy. (A) Force-induced displacement of a microtubule. Images from a time course (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043662#pone.0043662.s012" target="_blank">Videos S7</a>) have been background-subtracted. Frames are displayed at 30 s intervals. Microtubule contour is overlaid in blue. Scale bar is 3 µm. (B) 3-Dimensional imaging of curved microtubule using Parallax and TIRF microscopies. The microtubule displayed in (A, first frame) is plotted as a series of (x,y,z) coordinates. A pair of 2-dimensional TIRF images (A: 1 of pair) was used to calculate relative z-depth. Warmer colors are closer to the coverslip, as indicated by the calibration bar along the z-axis. For an animation of the 3-dimensional time course see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043662#pone.0043662.s013" target="_blank">Video S8</a>.</p

Time course parameters.

<p>Time course parameters are calculated from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043662#pone-0043662-g001" target="_blank">Figures 1</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043662#pone-0043662-g002" target="_blank">2</a>, and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043662#pone.0043662.s005" target="_blank">S5</a>.</p>a<p>Max fold intensity or density increase relative to baseline. CI, confidence interval.</p>b<p>Number of cells.</p

Vesicle movement prior to fusion with the plasma membrane.

<p>Vesicle movements prior to fusion with the plasma membrane were determined in 3T3-L1 adipocytes (n = 4 cells) transfected with mCherry-IRAP-pHluorin and stimulated with 100 nM insulin.</p>a<p>Vesicle movements were scored as unknown when high local vesicle density or low mCherry fluorescence intensity made determination of movement difficult.</p>b<p>Linear displacement of ∼1 µm or more was undetected prior to fusion of vesicle.</p

Insulin stimulation increases the intensity of HA-GLUT4-eGFP within the TIRF illumination zone.

<p>Adipocytes transfected with HA-GLUT4-eGFP were serum-starved prior to stimulation with 100 nM insulin at t = 0 min. Images were acquired using TIRF microscopy at 1 frame per 10 s. (A) Pseudocolor of HA-GLUT4-eGFP intensity before and after insulin stimulation. Elapsed time from insulin addition is indicated. Scale bar is 20 µm. The calibration bar at left indicates HA-GLUT4-GFP intensity with black and white the least and greatest intensities, respectively. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043662#pone.0043662.s006" target="_blank">Video S1</a>. (Inset) Brightfield image. Box is 27 µm×27 µm. (B) Time course of the increase in fluorescence intensity in response to insulin. Fold intensity increase relative to the average intensity prior to insulin addition was calculated for each cell. Plotted is the mean ±95% confidence interval (n = 13 cells). (Inset) Histogram of the fold intensity change at plateau.</p

Centromeres are maintained by fastening CENP-A to DNA and directing an arginine anchor-dependent nucleosome transition

Maintaining centromere identity relies upon the persistence of the epigenetic mark provided by the histone H3 variant, centromere protein A (CENP-A), but the molecular mechanisms that underlie its remarkable stability remain unclear. Here, we define the contributions of each of the three candidate CENP-A nucleosome-binding domains (two on CENP-C and one on CENP-N) to CENP-A stability using gene replacement and rapid protein degradation. Surprisingly, the most conserved domain, the CENP-C motif, is dispensable. Instead, the stability is conferred by the unfolded central domain of CENP-C and the folded N-terminal domain of CENP-N that becomes rigidified 1,000-fold upon crossbridging CENP-A and its adjacent nucleosomal DNA. Disrupting the 'arginine anchor' on CENP-C for the nucleosomal acidic patch disrupts the CENP-A nucleosome structural transition and removes CENP-A nucleosomes from centromeres. CENP-A nucleosome retention at centromeres requires a core centromeric nucleosome complex where CENP-C clamps down a stable nucleosome conformation and CENP-N fastens CENP-A to the DNA. Keywords: centromeres; supramolecular assembl