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| ImageStream images of the cell cycle phases in MEF cells. Representative fluorescence images of lamin A (green), tubulin (red), and chromatin (blue) in 5 different stages of the cell cycle, together with the brightfield (BF) image. Merged images show the lamin A, chromatin, and tubulin localization.
Source publication
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 c...
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Context 1
... measured thousands of MEF cells in order to follow the localization of lamin A during mitosis, and its colocalization relative to the chromatin and tubulin. Representative images of each cell cycle stage are shown in Figure 1. We present the brightfield image of the cell and three different fluorescent channels: lamin A in green, tubulin in red, and chromatin in blue, as well as a merged image of three of them, for the interphase and the four mitotic phases (prophase, metaphase, anaphase, and telophase). ...
Context 2
... one can see in Figure 1, in the early stages of mitosis, namely, prophase, metaphase, and anaphase, lamin A is distributed throughout the cytoplasm. However, 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. ...
Context 3
... to a similar number of cells with an imaging system, will take a much longer time, probably few days. It is important not to use synchronization that would makes it easier to find mitotic cells, but it was also shown to affect the biological state of the cells (Panet et al., 2015). Nevertheless, the method also has disadvantages, the major one arises from the fact that in order to measure in flow, the cells must be maintained in suspension which is not their natural adherent form. ...
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Citations
... The self-assembly of cytoskeletal filaments is crucial for many cellular functions, including wound healing [1] and cell division [2]. The growth kinetics of these filaments strongly influences the morphology of the networks they form, from bundled to entangled structures [3][4][5][6][7]. ...
The kinetics of the assembly of semiflexible filaments through end-to-end annealing is key to the structure of the cytoskeleton, but is not understood. We analyze this problem through scaling theory and simulations, and uncover a regime where filaments’ ends find each other through bending fluctuations without the need for the whole filament to diffuse. This results in a very substantial speedup of assembly in physiological regimes, and could help with understanding the dynamics of actin and intermediate filaments in biological processes such as wound healing and cell division.
... In addition, the compression-induced lamin A/C de ciency was completely rescued after 6 h according to Western blot analysis (Fig. 1a&b). Numerous evidence has revealed that lamin A/C works dynamically in acclimating external environment and regulating chromatin accessibility in the cell cycle [22,55,56]. The lamin A/C level is proportional to nuclear stiffness, and it can be enhanced by some external stimulations, such as a rigid extracellular matrix [22,57]. ...
Cellular mechanosensation is a complex physiological process coupling alterations in the external environment and cellular behaviors. Over the past decade, the role of the nucleus in mechanosensation has gained increasing attention. Our research found that lamin A/C, a component of the nuclear envelope, plays a dual role in the mechanosensation of macrophages in response to compressive force. Our findings indicated that hydrostatic compressive force downregulated lamin A/C protein via the cytoskeleton. Consequently, this lamin A/C deficiency enhanced compressive-force-induced inflammatory cytokines secretion and proliferative impairment. Unexpectedly, lamin A deficiency also inhibits compressive force-induced DNA damage and interferon regulatory factor 4 (IRF4) up-regulation. Our findings suggest that lamin A/C is involved in multiple mechanosensation mechanisms. Mechanistically, lamin A/C deficiency augments nuclear permeability, facilitates the activation of yes-associated protein 1 (YAP1) and promotes force-induced nuclear translocation of YAP1. These mechanisms have been validated to favor mechanosensation. Conversely, we also found that lamin A/C deficiency led to detachment of components of linker of nucleoskeleton and cytoskeleton (LINC) complex, which impeded intracellular mechanotransmission. In summary, lamin A/C can promote some responses of macrophages to mechanical compression but inhibits others. It is involved in two distinct mechanisms: enhancing nuclear permeability to transcription factors and impairing mechanotransmission by disrupting the LINC complex's connection to the nucleus.
... The A-type lamins, lamin A/C, are found in the nuclear periphery and nuclear interior, while the B-type lamins, lamin B1 and B2, are concentrated in the nuclear periphery [47]. The function of the nuclear lamina is to provide a structural (mechanical) framework for the nucleus [48], and assist other functions in the nucleus morphology, such as nucleoplasmic reticulum formation [49,50], support chromatin organization by defining territories within the nucleus and directly tethering LADs to the nuclear envelope [51][52][53][54], support the mitosis cycle by regulating mitotic spindle assembly and positioning [55,56], participate in DNA replication with focal replication regions in early S-phase [57,58], and regulate gene transcription through interactions with transcription factors, such as barrier-to-autointegration factor (BAF), that directly binds to DNA and it is involved in transcription control at an epigenetic level [59][60][61]. ...
Cardiac muscle contraction is distinct from the contraction of other muscle types. The heart continuously undergoes contraction–relaxation cycles throughout an animal’s lifespan. It must respond to constantly varying physical and energetic burdens over the short term on a beat-to-beat basis and relies on different mechanisms over the long term. Muscle contractility is based on actin and myosin interactions that are regulated by cytoplasmic calcium ions. Genetic variants of sarcomeric proteins can lead to the pathophysiological development of cardiac dysfunction. The sarcomere is physically connected to other cytoskeletal components. Actin filaments, microtubules and desmin proteins are responsible for these interactions. Therefore, mechanical as well as biochemical signals from sarcomeric contractions are transmitted to and sensed by other parts of the cardiomyocyte, particularly the nucleus which can respond to these stimuli. Proteins anchored to the nuclear envelope display a broad response which remodels the structure of the nucleus. In this review, we examine the central aspects of mechanotransduction in the cardiomyocyte where the transmission of mechanical signals to the nucleus can result in changes in gene expression and nucleus morphology. The correlation of nucleus sensing and dysfunction of sarcomeric proteins may assist the understanding of a wide range of functional responses in the progress of cardiomyopathic diseases.
... Although lamins are stable proteins with longer cellular half-lives, 76 the disassembly and reassembly of lamins in the nuclear lamina during cell division is an essential part of the cell cycle. 77 In addition, the nuclear lamina also plays a role in regulating gene expression 78 through chromatin interactions 79,80 and maintaining cytoskeletal dynamics in the cell. 81 Although structurally robust, lamin function and localization are regulated by various post-translational modifications (PTMs). ...
Laminopathies are a group of rare genetic disorders with heterogeneous clinical phenotypes such as premature aging, cardiomyopathy, lipodystrophy, muscular dystrophy, microcephaly, epilepsy, and so on. The cellular phenomena associated with laminopathy invariably show disruption of nucleoskeleton of lamina due to deregulated expression, localization, function, and interaction of mutant lamin proteins. Impaired spatial and temporal tethering of lamin proteins to the lamina or nucleoplasmic aggregation of lamins are the primary molecular events that can trigger nuclear proteotoxicity by modulating differential protein–protein interactions, sequestering quality control proteins, and initiating a cascade of abnormal post‐translational modifications. Clearly, laminopathic cells exhibit moderate to high nuclear proteotoxicity, raising the question of whether an imbalance in nuclear proteostasis is involved in laminopathic diseases, particularly in diseases of early aging such as HGPS and laminopathy‐associated premature aging. Here, we review nuclear proteostasis and its deregulation in the context of lamin proteins and laminopathies.
... b. Cell division: using the DNA staining, you can determine the stages of cell division by DNA morphology (examples can be seen in (Filby et al., 2011); (Wortzel et al., 2015); (Vivante et al., 2021)), however adjustments may be needed according to particular cell type. i. Gate for G2/M based on DNA staining intensity. ...
The Golgi apparatus is subjected to fragmentation under several cellular processes such as mitosis. Here we describe two complementary approaches to analyze different Golgi morphological changes during its mitotic fragmentation, using classical immunofluorescence and imaging flow cytometry. Although fluorescent microscopy provides information on the exact Golgi architecture in distinct cells, the imaging flow cytometry combines the morphological data with the high-throughput quantification of flow cytometry. Taken together, both approaches provide robust and significant unbiased data analysis.
For complete details on the use and execution of this protocol, please refer to Wortzel et al. (2021).
... However, during telophase, when lamin A and lamin B1 are already colocalized at the nascent NE, lamin C was solely nucleoplasmic, with no detectable concentration at the NE ( Fig. 3 and Additional file 1: Fig. S11). This telophase association of lamin A with the NE has recently been confirmed in a high throughput imaging screen with a lamin A specific antibody [55]. We verified these results using antibodies specific for each of the lamin isoforms (lamin B1, lamin A, and lamin C; Additional file 1: Fig. S12). ...
Background
The dynamic 3D organization of the genome is central to gene regulation and development. The nuclear lamina influences genome organization through the tethering of lamina-associated domains (LADs) to the nuclear periphery. Evidence suggests that lamins A and C are the predominant lamins involved in the peripheral association of LADs, potentially serving different roles.
Results
Here, we examine chromosome architecture in mouse cells in which lamin A or lamin C are downregulated. We find that lamin C, and not lamin A, is required for the 3D organization of LADs and overall chromosome organization. Striking differences in localization are present as cells exit mitosis and persist through early G1 and are linked to differential phosphorylation. Whereas lamin A associates with the nascent nuclear envelope (NE) during telophase, lamin C remains in the interior, surrounding globular LAD aggregates enriched on euchromatic regions. Lamin C association with the NE is delayed until several hours into G1 and correlates temporally and spatially with the post-mitotic NE association of LADs. Post-mitotic LAD association with the NE, and global 3D genome organization, is perturbed only in cells depleted of lamin C, and not lamin A.
Conclusions
Lamin C regulates LAD dynamics during exit from mitosis and is a key regulator of genome organization in mammalian cells. This reveals an unexpectedly central role for lamin C in genome organization, including inter-chromosomal LAD-LAD segregation and LAD scaffolding at the NE, raising intriguing questions about the individual and overlapping roles of lamin A/C in cellular function and disease.
Chromobodies made of nanobodies fused to fluorescent proteins are powerful tools for targeting and tracing intracellular proteins in living cells. Typically, this is achieved by transfecting plasmids encoding the chromobodies. However, an excess of unbound chromobody relative to the endogenous antigen can result in high background fluorescence in live cell imaging. Here, we overcome this problem by using mRNA encoding chromobodies. Our approach allows one to precisely control the amount of chromobody expressed inside the cell by adjusting the amount of transfected mRNA. To challenge our method, we evaluate three chromobodies targeting intracellular proteins of different abundance and cellular localization, namely lamin A/C, Dnmt1 and actin. We demonstrate that the expression of chromobodies in living cells by transfection of tuned amounts of the corresponding mRNAs allows the accurate tracking of their cellular targets by time‐lapse fluorescence microscopy.
Eukaryotic interphase chromosomes maintain a three-dimensional structure within the nucleus and undergo fluctuations. It has been reported that such dynamics are involved in transcription, replication, and DNA repair. However, the analysis of chromosomal dynamics has been limited to high-throughput chromosome conformation capture data, which records the contact frequencies between chromosomal regions and lack direct information about the dynamic. Herein, we investigated chromosome fluctuations as polymers based on experimental data from sequential fluorescence in situ hybridization (seqFISH)+ using a multiomics methodology. To describe the principal modes of chromosome fluctuations, we applied principal component analysis to the three-dimensional structure information of single chromosomes in 446 mouse embryonic stem cells (mESCs) obtained from seqFISH+ data analysis for spatial genomics and nuclear factors (histone marks, repeat DNAs, and nuclear compartments). We found that chromosome fluctuations exhibit both isotropic and anisotropic modes. The properties of anisotropy in chromosome fluctuation vary among chromosomes and appear to depend on the interaction between repeat DNAs on the chromosomes and nuclear factors. Furthermore, our principal component analysis revealed anisotropic chromosome fluctuations before and after the mitotic phase, specifically when chromosomes adopt a spindle-like shape. This result suggests the potential involvement of anisotropic chromosomal fluctuations in the transition of nuclear organization during the cell cycle. Our results represent the first study to elucidate the dynamics of chromosomes as polymers based on real multiomics data.
