Abstract
Lamin proteins play an essential role in maintaining the nuclear organization and integrity; and lamin A, in particular, plays a major role in the whole volume of the nuclear interior. Although the nucleus is highly organized, it is rather dynamic, it affects crucial nuclear processes and its organization must change as cells progress through the cell cycle. Although many aspects of these changes are already known, the role of lamin A during nuclear assembly and disassembly as well as its underlying mechanisms remains controversial. Here we used live cells imaging and Continuous Photobleaching (CP) method to shed light on the dynamics and mechanisms of lamin A during the cell cycle, combined with imaging flow cytometry measurements, which provides the high-throughput capabilities of flow cytometry with single-cell imaging. As a major analysis tool, we used spatial correlation algorithm for allocating the distribution of lamin A, chromatin and tubulin, as well as their mutual colocalization. Furthermore, we analyzed the distribution of lamin A along the nuclear lamina and in the nucleus interior during the cell cycle. Our results indicate that at the beginning of the cell division that include prophase, metaphase and anaphase, lamin A is distributed throughout the cytoplasm and its concentration in the chromosomal regions is reduced, whereas the spatial correlation between lamin A and tubulin is increased. It implies that lamin A also disassembled in the whole cellular volume. At the telophase and early G1, lamin A is concentrated in the whole volume of the newly formed nuclei of the daughter cells and it assembles to the lamina. We also explored the functional aspects of lamin A during the cell cycle and its binding to the chromatin versus the freely diffusion form. We found that the fraction of the bound proteins of lamin A in the S phase increased, relative to the G1 phase, which means that during replication, the concentration of lamin A on the chromatin increases. All these results shed light on the function of lamin A throughout the cell cycle.
Highlights
In recent years, a growing number of studies have revealed the hierarchical organization of chromatin in the cell nucleus and have highlighted its importance for maintaining adequate cellular functions throughout the cell cycle
We measured thousands of Mouse Embryonic Fibroblast (MEF) cells in order to follow the localization of lamin A during mitosis, and its colocalization relative to the chromatin and tubulin
lamin A and anti-tubulin primary antibodies (Lamin A)’s distribution is not necessarily uniform because it is gradually reduced throughout mitosis in the region containing the chromosomes and it is increased in the tubulin region
Summary
A growing number of studies have revealed the hierarchical organization of chromatin in the cell nucleus and have highlighted its importance for maintaining adequate cellular functions throughout the cell cycle. The lamin proteins, which consist of lamin A/C, lamin B1, and lamin B2, play an essential role in maintaining the nuclear organization. The nuclear lamins are intermediate filament (IF) proteins (Dechat et al, 2010) that self-assemble into higher-. Lamin A Dynamics During the Cell-Cycle order structures. The lamin dimers associate in a head-to-tail fashion and interact in an antiparallel fashion to form filaments (Heitlinger et al, 1992). The most prominent structure assembled from the lamins is the nuclear lamina, which is found at the inner face of the nuclear envelope membrane (Gerace et al, 1978; Krohne et al, 1978). Lamins, and especially lamin A, are located at the nuclear periphery and in the nuclear interior (Broers et al, 1999)
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