核染色体活动的首个图像证据

yujian623

细胞是高度组织化的，不同的区域有不同的功能，分子马达穿梭其间。现在，美国伊利诺斯州大学和Urbana-Champaign的研究人员从生活细胞获得了细胞核中进行的组织和运输的第一个图像证据。研究的结果公布在4月17日的Current Biology杂志上。

    已经知道，活动的基因主要定位在细胞核的中心区域，而不活动的基因则定位在外围。但是，研究人员却苦于无法追踪细胞核内的染色体活动或确定染色体的位置是否是随机扩散的结果。

    在这项新的研究中，美国的研究人员证实细胞核中的染色体依赖肌球蛋白和肌动蛋白进行定向的长距离的运动。

    由于染色体运动对光及其敏感，因此要发明一种能够观察细胞核运动的系统变得非常困难。一接触光，染色体就可能不在运动。通过长期广泛的研究，美国的研究人员发明了一种能够在不影响这种染色体运动的情况下获得活动影像的方法。利用这种系统，研究人员分析了一种通常呆在细胞核外围并处于非活动状态的染色体，并且发现她在接受到一个活化信号时会向着细胞核内部运动。

    这种在活化后的染色体运动不同于之前在细胞核中观察到的迅速但短距离的扩散运动。观察结果很明显地表明，这是个需要马达的定向运动，因为染色体几乎向着细胞核进行着直线运动。染色体能够运动若干分子甚至若干小时。

    当研究人员将一种突变形式的肌球蛋白引入到细胞核中时，这种运动就缓慢下来；当引入一种不能形式纤丝的突变形式的肌动蛋白时，就会政治这种运动。另外，当加入能抑制肌动蛋白/肌球蛋白的药物时，这种染色体运动就被完全终止。这些实验强有力地表明肌动蛋白和肌球蛋白与染色体的运动有关。





Figure 1. VP16 Induces Chromosome Repositioning from Nuclear Periphery to Interior

(A) EGFP-lac repressor protein with three copies of FKBP12 binds nonfluorescent FRB-VP16 after rapamycin addition.

(B) Percentage of log phase cells with tagged chromosome spot less than 0.5 μm from nuclear periphery at different times after rapamycin addition (bars represent standard deviation of the mean based on three experiments).

(C–H) Percentage of peripheral (black) versus interior (gray) sites.

(C) Similar redistribution of chromosome site 2 hr after VP16 targeting in cells blocked in late G1/early S.

(D) The acidic activation domain from p65, an endogenous protein, produces similar spot redistribution as seen for the VP16 AAD after targeting induced by rapamycin addition.

(E) Redistribution toward cell interior occurs after targeting an acidic peptide capable of decondensing large-scale chromatin structure but with no transcriptional activity.

(F) Redistribution of chromosome site after transcription inhibition by DRB.

(G and H) Spot redistribution 2 hr after adding rapamycin is dependent on illumination conditions. Data points are from 100–200 cells (C–H) or 300 cells (B).


Figure 2. Live Cell Imaging Reveals Long-Range Spot Movements

(A) Chromosome site (arrowheads) has moved toward nuclear interior 2 hr after rapamycin addition.

(B) Scatter plot showing net radial movement of spots between 0 and 2 hr after rapamycin addition in cells expressing FRB-VP16 (black diamonds) or control cells not expressing FRB-VP16 (open circles). x axis is starting distance from nuclear periphery, y axis is net radial distance moved toward (positive) or away (negative) from nuclear interior.

(C) Spot undergoing long-range motion: numbers show time in minutes after rapamycin addition followed by optical section number (out of 21) from z-stack (0.075 μm focus step). Movement occurs roughly perpendicular to nuclear envelope.

(D) Spot movement occurs without significant change in focus or nuclear shape during period of rapid motion. Optical sections ∼0.75 μm below (top) or above (bottom) spot focus (middle) are shown from this period of rapid spot movement. (Upper right, optical section number, 0.075 μm focus step; middle row, panel lower left, time in minutes after rapamycin addition.)

(E) Second example showing spot movement, with minimal changes in focus, from periphery toward the nuclear interior. Numbers shown are as in (C). Scale bars equal 5 μm.








Figure 3. Long-Range, Rapid Movements Show Curvilinear Trajectories

From each time point, the single optical section for which the spot was in focus was extracted from the 3D data stack. A crosscorrelation approach was used to align these 2D images from adjacent time points to correct for nuclear translation and rotation and small changes in nuclear shape, forming an image stack where each section now corresponds to a different time point. This image stack was then projected to yield the spot trajectory over time.

(A) Linear trajectory (60′–67′) is followed by longer period of localized, short-range motion (68′–80′). Time refers to minutes after rapamycin addition.

(B and C) Trajectories of a cell not showing long-range motion after rapamycin addition (B) and a control cell (no rapamycin) (C) showing limited, apparently random movements confined within small region. The radius of confinement appears smaller for the control cells versus cells exposed to rapamycin that did not show long-range spot movements (see also Figure 4).

(D–M) Trajectories from cells showing long-range spot movements.

(E) Corresponds to 60′–64′, all the rest are 60′–79′. Scale bars equal 5 μm.







Figure 4. Two Qualitatively Different Mobility Regimes—Rapid, Long-Range Mobility Is Distinct from Short-Range, Localized Mobility

(A) Distance from nuclear periphery versus time—during short periods of rapid, long-range motion, roughly constant velocities (i.e., linear plots) are observed. Red plot corresponds to cell shown in Figures 2C, 2D, 3A, and 3D orange plot corresponds to cell shown in Figures 2E and 3G.

(B) Relative changes in focus for same cells shown in (A).

(C) Distance from nuclear periphery versus time for spots from cells treated with rapamycin but not showing long-range movements. Fluctuations in distance from nuclear envelope appear several times larger than observed in control cells (no rapamycin), which show only small position fluctuations and no persistent, unidirectional, long-range movements (D).

(E and F) Mean square distance from periphery versus time lag shows qualitative differences in mobility. (E) Averages from 14 trajectories showing long-range motion (blue), including 10 curvilinear trajectories from aligned data sets (red), versus 29 spot trajectories (orange in [E] and [F]), which did not show long-range motion after rapamycin addition, and 30 control trajectories (green in [E] and [F]).



Figure 5. Functional Analysis of Actin and Myosin in Spot Migration

(A) Recruitment of nuclear myosin I (red) to A03 amplified chromosome region (homogeneous staining region, HSR) containing lac operator sites (arrows) after expression of EGFP-lac repressor-VP16 AAD (green). No recruitment was seen after expression of EGFP-lac repressor (green). Scale bar equals 5 μm.

(B–F) Percentage of cells with peripheral chromosome location (averages from two [B–E] or three [F] experiments, 100 cells each).

(B) 3 hr delay in spot redistribution in cells expressing E407 NMI relative to control, nontransfected cells.

(C) No delay in cells expressing NMI with myopathy loop deletion.

(D) BDM blocks spot redistribution.

(E) Expression of mRFP-NLS-G13R mutant actin defective in polymerization blocks spot movement at all times.

(F) S14C actin mutant, favoring F-actin polymerization, accelerates spot redistribution (black) relative to wild-type actin (gray).