It is also possible that formation of 10 nm filaments depends on the presence of membranes that are absent from the in vitro systems. Instead, most lamins form paracrystalline arrays in vitro , which display a unique axial repeat pattern [ 33 ]. Heterotypic lamin dimers can interact to form mixed isoform polymers in vitro [ 34 ].
However, it is unclear whether in vivo individual filaments contain both types of lamins within the same filament or whether lamins segregate into distinct filaments. Following mitosis, lamin A incorporates into the membrane after lamin B, suggesting that nuclear IFs are homotypic [ 35 ]. Nevertheless, this does not exclude the possibility of eventual reorganization of the nascent lamina or integration of heterotypic interactions, as suggested by high-resolution images of a mammalian somatic lamina that demonstrate domains of segregation and domains of overlap juxtaposed within the same nucleus [ 24 ].
Lamins are post-translationally modified in several ways. Almost all lamins contain a CAAX box at the carboxyl terminus, which serves as a substrate for post-translational farnesylation. This is achieved in three successive steps starting with the isoprenylation of the cysteine, followed by the proteolytic cleavage of the AAX motif and finally the carboxy-methylation of the farnesylated cysteine. The hydrophobic farnesyl moiety is thought to facilitate localization to and retention at the nuclear envelope.
Nevertheless, carboxy-terminal farnesylation is not absolutely required for lamina localization because lamin C does not contain a CAAX box but does localize to the nuclear envelope. Once localized to the nuclear envelope, lamin A is processed further by the proteolytic removal of the carboxy-terminal 18 amino acids, including the farnesyl group, whereas B-type lamins retain the farnesyl moiety in their mature form [ 33 ].
Lamins are phosphorylated by multiple kinases and contain many conserved phosphorylation sites, with more than 30 known sites in human A-type lamins alone [ 36 ]. The mitotic kinase CDC2 induces lamin disassembly by phosphorylating conserved residues within the head and coiled-coil 2B domains [ 31 ]. Phosphorylation of Thr19, Ser22 and Ser causes depolymerization of lamin filaments in mitosis and meiosis. PKA phosphorylation sites are highly conserved and when phosphorylated inhibit lamin polymerization [ 37 ].
In addition, phosphorylation by PKC is known to regulate lamin uptake into the nucleus [ 38 ]. Lamin A also has two sumoylation consensus sites, one within the rod domain and another within the tail domain.
Substitutions EG and EK within the rod domain disrupt the canonical SUMO E2 site, resulting in lower levels of sumoylated lamin in vivo and altered subnuclear localization [ 39 ]. B-type lamins are constitutively expressed in most cell types, whereas A-type lamins are developmentally regulated, being predominantly expressed in most differentiated cell types [ 40 ].
However, certain cells of the hematopoietic system do not express A-type lamins even when fully differentiated [ 41 ]. In mammalian germ cells and pronuclei, expression is limited to the two atypical lamin isoforms lamin B3 and C2, respectively, which are detected only in germ cells but are otherwise not expressed [ 18 , 42 ]. A- and B-type lamins are also found in fertilized mammalian eggs until 2 to 4 cleavage divisions, when A-type lamins are no longer detected.
As embryogenesis proceeds, A-type lamins are again detected on day 8 to 9 in extra-embryonic tissues and on day 12 in the embryo itself [ 41 , 43 ]. Mouse and human embryonic stem cells express lamins B1 and B2 but not lamins A or C. Interspecies differences in lamin expression have also been noted: lamin A is highly expressed in circulating erythrocytes in Gallus gallus chicken but is absent from the same cell type in amphibians [ 44 , 45 ].
Because A-type lamins usually appear after cellular differentiation initiates, it is thought that A-type lamins facilitate 'locking-in' of the differentiated state [ 41 ]. As expected from their incorporation into the lamina, a large portion of cellular lamin is found in an insoluble pool at the nuclear rim Figure 4. There is also a pool of A-type lamins within the nucleoplasm, which is distinct from peripheral lamin A, in that it is probably not polymerized and is more soluble [ 46 ].
The assembly state, fraction, purpose and function of the nucleoplasmic lamin A are still unknown [ 47 , 48 ]. Similarly, B-type lamins are also present in the nucleoplasm. In vivo dynamics of lamins in interphase nuclei have been studied by various techniques, including fluorescence recovery after photobleaching and fluorescence correlation spectroscopy [ 24 , 35 , 49 — 51 ]. In contrast to cytoplasmic IF proteins, which seem very dynamic, nuclear lamins are relatively stable once integrated into the nuclear lamina [ 49 ].
Cell-cycle-related changes to lamin protein dynamics have also been reported. For instance, lamina-associated lamin B1 associates with the lamina with a half-life of about 10 minutes during the initial stages of G1, which increases to about 2 hours in a later part of G1 [ 35 ]. Nuclear lamins: localization at the nuclear periphery and within the nucleoplasm.
Lamins have pivotal roles in nuclear reassembly after cell division. During mitosis, when the nuclear envelope breaks down and the lamina disassembles, A-type lamins are solubilized and distributed throughout the cytoplasm, whereas B-type lamins maintain close associations with the nuclear membrane.
The differences in membrane attachment during mitosis are attributed to whether the lamin protein is farnesylated. Mature lamin B retains its farnesylation moiety, which anchors B-type lamins to the membrane during mitosis, whereas the farnesylation moiety is removed from lamin A, rendering it more soluble [ 52 ]. To facilitate disassembly of the lamina, lamins are phosphorylated by PKC and are dephosphorylated by type 1 protein phosphatase during reassembly [ 53 ].
When a nascent nuclear envelope forms around condensed chromosomes, A-type lamins are imported into the nucleus along with additional B-type lamins [ 35 , 54 ].
Lamins are early targets for caspase degradation in cells undergoing apoptosis [ 55 , 56 ]. Caspase-6 and caspase-3 are the major proteases responsible for A- and B-type lamin degradation [ 57 ]. At the onset of apoptosis, before detectable DNA cleavage or chromatin condensation occurs, lamins are cleaved at caspase recognition sites located within the L12 linker region and expression of uncleavable mutant lamin protein delays the onset of apoptosis [ 58 ].
Time-lapse experiments of green-fluorescent-protein-tagged lamins suggest that A- and B-type lamins have different dynamics following their initial cleavage [ 59 ]. A-type lamins are thought to rapidly translocate to the nucleoplasm and cytoplasm, whereas B-type lamins remain at the nuclear periphery.
From their primary sequence and their grouping within the IF superfamily, the lamins were originally hypothesized to provide mechanical support for the nucleus, conceivably as tensegrity elements that specify nuclear morphology and resistance to deformation [ 22 ]. In support of this, studies using Xenopus nuclear assembly systems show that cell-free extracts depleted of lamins assemble small and fragile nuclei [ 60 ].
Additional studies have demonstrated that human cells expressing a variety of lamin mutations often show a range of nuclear morphological phenotypes [ 62 ]. The extent of B-type lamin involvement in nuclear mechanics is not as well understood because loss of lamin B1 from murine embryonic fibroblasts MEFs causes nuclear blebbing, but does not seem to affect mechanical properties [ 66 ]. In addition to A- and B-type lamin interactions, numerous functionally diverse proteins are known to interact with the nuclear lamins, including retinoblastoma 1, c-Fos, thymopoietin LAP2 and emerin Figure 5.
Lamins are part of a nuclear framework supporting multi-protein complexes involved in several nuclear functions. The majority of lamin-interacting proteins identified so far come from studies focused on A-type lamins. More than 30 direct and more than indirect interactions have been identified using various proteomics-based studies [ 67 , 68 ]. The extensive list of interaction partners further supports the notion of the lamina functioning as an intranuclear platform.
The nature and function of each interaction probably varies, possibly in a tissue-specific manner. Functions of the nuclear lamina. A cartoon representation of the nuclear lamina, highlighting four key functions. P, phosphate. Nuclei are mechanically linked to the cytoskeleton through lamin-interacting proteins that span the nuclear envelope Figure 5. This linker of nucleoskeleton and cytoskeleton LINC complex consists of lamin-interacting proteins SUN1 or SUN2, which span the inner nuclear membrane where they, in turn, interact with a member of the nesprin family of proteins in the luminal space [ 70 ].
Nesprins span the outer nuclear membrane, where they associate in the cytoplasm with various cytoskeletal elements. The LINC complex has been implicated in serving functions important for nuclear migration, positioning, morphology and mechanics [ 71 , 72 ]. Lamins are global regulators of chromatin Figure 5. Transcriptionally silent regions of the genome, such as centromeres, telomeres and the inactive X chromosome, are preferentially positioned at the nuclear lamina [ 73 , 74 ].
Global heterochromatic changes induced by lamin perturbation are often mirrored by altered levels of chromatin-associated epigenetic histone marks; for example, decreased levels of the heterochromatin markers histone H3 lysine 9 trimethylation H3K9me3 and H3K27me3 and increased levels of H4K20me3.
Ectopic lamin expression also influences chromatin organization and associated histone marks; for example, hypermethylated H3K4, a mark of active genes, decreases following overexpression of wild-type lamin A in C2C12 myoblasts [ 81 ]. Lamins have at least two chromatin-binding regions. One chromatin interaction domain is located in the tail region between the end of the rod domain and the Ig domain, and the other is within the rod domain [ 82 , 83 ].
Genome-wide mapping techniques have identified genome regions that preferentially associate with lamins, known as lamin-A-associated domains LADs [ 86 ]. These domains are generally gene-poor and are proposed to represent a repressive chromatin environment.
Recent studies have hinted at a role for lamins in DNA repair. Using the CP method on MF cells, we explored the ratio of the free fraction to the bound fraction of lamin A protein in the G1 and S phases thereby quantifying the localization level of the protein at each phase Figure 6. The red and blue shaded areas above and below each curve refer to the STD of the measurements. Therefore, our results indicate that the fraction of the bound proteins of lamin A in the S phase increased, relative to the G1 phase.
The localization and dynamics of lamin A during interphase have been previously studied Bronshtein et al. It was shown that lamin A is required for the 3D organization of chromosomes and for maintaining the chromosome territories in the cell nucleus. However, during mitosis and the nuclear lamina assembly process at the end of mitosis, the localization and function of lamin A remain to be elucidated. For measuring 3D organization during mitosis, we used multispectral imaging flow cytometry.
The method has the advantage of measuring thousands of cells in a 2—3 h experiment. Getting 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.
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.
It was shown before that cells that are being disassociated from the slide go throw biomechanical changes and that lamin A is phosphorylated Buxboim et al.
However, to overcome that in our study, the fixation was performed a short time after disassociation less than 10 min. Furthermore, by observing the cells from our results, we note that cells that are in interphase have a well distinct lamina which assures that a significant percentage of lamin A proteins is not phosphorylated, otherwise the lamina should be completely disassembled.
It can be important to repeat similar measurements with a high-throughput system that will allow to measure adherent cells. We found that during the early stages of mitosis, namely, prophase, metaphase, and anaphase, lamin A is distributed throughout the cytoplasm, but not necessarily uniformly.
Lamin A is gradually reduced in the region containing the chromosomes and its concentration increases in the tubulin region. The distribution of lamin A relative to the chromatin, indicates that not only lamin A disassembles from the chromatin during mitosis, maybe due to the phosphorylation of lamin A, it also avoids the region of the chromatin for proper mitosis.
The distribution of lamin A relative to tubulin, indicates their mutual affect during mitosis. However, other studies have shown that lamin A only gradually becomes incorporated into the peripheral lamina during the first few hours of early G1 of the cell cycle Moir et al.
These differences could be explained by variations between different cell types. Our previous studies suggested that lamin A proteins form chromatin cross-links during interphase and therefore maintain the chromatin organization.
However, the mechanism and the interactions between lamin A and chromatin during the cell cycle raise interesting open questions. During mitosis prophase, metaphase, and anaphase , lamin A is distributed throughout the cytoplasm in a freely diffusive manner, and it is reassociated with chromatin during early G1 stage Moir et al.
During interphase the G1, S, and G2 phases , some of the proteins freely diffuse, whereas others are bound Bronshtein et al. According to our CP results, in the S phase the fraction of bound lamin A proteins is slightly increased, relative to the G1 phase.
The fraction of the bound lamin A that we found in both phases is smaller than previously reported Bronshtein et al. The higher fraction of bound proteins during the S phase is most likely due to the larger amount of chromatin in the nucleus that requires more bound lamin A for maintaining its order.
This is in agreement with another study, which explored the lamina-associated domains LADs during the cell cycle, and showed that S phase chromatin is characterized by transiently increased lamina interactions van Schaik et al. Previously, a study of the dynamic properties of chromatin in cells that do not express lamin A, did not find significant differences between the chromatin dynamics in G1, S, and G2 phases Bronshtein et al.
However, depletion of lamin A results in a significant increase in the chromatin dynamics in all cell cycle stages; therefore, this small difference in the bound fraction of the proteins between the G1 and S phase is neglected here. Taken together, Lamin A proteins form chromatin cross-links during interphase in the whole nuclear volume, which are widespread throughout the genome.
Together with the chromatin binding to the lamina, these mechanisms are crucial for chromatin organization. The number and density of the cross-links in the nuclear interior is increased during DNA replication, as it is necessary to maintain the order of a double amount of chromatin in this phase. At the beginning of mitosis, lamin A phosphorylation leads to disassembly of the lamina from the chromatin. Our findings that during mitosis there is less lamin A in the chromatin regions suggests that phosphorylation also leads to the disassembly of lamin A in the whole cellular volume.
This finding, which correlates with few of the previous publications Bronshtein et al. Lamin A may have an effect on the cellular function that is not related directly to the chromatin organization; this is an interesting question that should be studied in the future. For future work, it will be interesting to continue study the distribution of other nuclear related proteins, such as lamin B, BAF and emerin.
In this study we combined single cell imaging with the high-throughput capabilities of conventional flow cytometry; this allows us to use asynchronously dividing cells for more natural and reliable results.
We explored the localization and interaction between lamin A and chromatin during the cell cycle phases, in order to shed light on the mechanism and dynamics of lamin A during the cell cycle and to reveal more details about this controversial issue. Our findings emphasize the significance of lamin A and chromatin interactions in the nucleus interior throughout the cell cycle interphase and mitosis for maintaining cell organization and function.
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. This study was designed by AV and YG who provided conceptual and technical guidance for the project. AV performed the measurements, planned the analytical tools, and analyzed the data. IS performed the ImageStream measurements, took part in the analysis, and provided useful comments. The manuscript was written by AV and YG. Grosskopf grant for Generalized dynamic measurements in live cells at Bar Ilan University.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers.
Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. We thank E. Panet and R. Lahmi from the A. Tzur lab Bar Ilan University for fruitful guidance and discussions. Al-Saaidi, R.
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APS can also cause similar abnormalities in bone development and fat distribution as mandibuloacral dysplasia, although they are typically milder in APS. Mutations in the LMNA gene have also been identified in newborns with a disorder called lethal restrictive dermopathy. Infants with this disorder have tight, rigid skin; underdeveloped lungs; and other abnormalities. They do not usually survive past the first week of life. Researchers have not determined how mutations in the LMNA gene result in this diverse group of disorders, but the multiple roles of the nuclear lamina in cells may help explain the wide variety of signs and symptoms.
Genetics Home Reference has merged with MedlinePlus. Learn more. The information on this site should not be used as a substitute for professional medical care or advice. Contact a health care provider if you have questions about your health. From Genetics Home Reference. Emery-Dreifuss muscular dystrophy More than mutations in the LMNA gene have been identified in people with Emery-Dreifuss muscular dystrophy, a condition that affects muscles used for movement skeletal muscles and the heart cardiac muscle.
More About This Health Condition. Familial partial lipodystrophy Several mutations in the LMNA gene have been found to cause familial partial lipodystrophy type 2 also known as familial partial lipodystrophy, Dunnigan type , a rare condition characterized by an abnormal distribution of fatty adipose tissue in the body. Hutchinson-Gilford progeria syndrome A specific mutation in the LMNA gene has been found in most patients with Hutchinson-Gilford progeria syndrome, which is a condition that causes the dramatic, rapid appearance of aging beginning in childhood.
Arrhythmogenic right ventricular cardiomyopathy MedlinePlus Genetics provides information about Arrhythmogenic right ventricular cardiomyopathy More About This Health Condition.
LMNA-linked lipodystrophies: from altered fat distribution to cellular alterations. Biochem Soc Trans. Emery-Dreifuss muscular dystrophy, laminopathies, and other nuclear envelopathies. Handb Clin Neurol. Muscle Nerve. Epub Jun Overlapping syndromes in laminopathies: a meta-analysis of the reported literature. Acta Myol.
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