Laminate

Lamines belong to a family of proteins that are responsible for maintaining the structure and function of the cell nucleus. Mutations in genes that code for lamines (LMNA/LMNB gene; > 200 mutations are known so far) can make the cell nucleus unstable and lead to the development of so-called laminopathies. This group of diseases, which includes muscular dystrophies, manifests itself shortly after birth and often leads to premature death. The development of therapeutic strategies is difficult because the mechanisms at the molecular level are largely unexplained.

Laminates are evolutionarily highly conserved. They are specific to animals. Plants or eukaryotic unicellular organisms do not express laminae. The individual lamina proteins are involved in nuclear stability, chromatin structure and gene expression. Some lamines play an important role in the reorganization of chromatin during meiosis. They can be subdivided according to their structural and biochemical properties and their behaviour during mitosis into:

• Type A Laminates
• and
• Type B laminates (Gruenbaum et al. 2003; Gruenbaum et al. 2005).

During mitosis, type A laminae are distributed as soluble proteins in the cytoplasm. During development, type B laminae are found in all stages and all cell types. The cysteine is farnesylated shortly after translation in the cytoplasm, whereupon the last three amino acids are split off. This is followed by methylation of the cysteine (Moir et al., 1995). The carboxy terminus, which is very hydrophobic due to this modification, finally interacts with the nuclear membrane. Even during mitosis, the type B-lamines remain associated with the inner nuclear membrane via the CxxM motif.

7 Lamin isoforms are known in vertebrates, which are produced by alternative splicing. These are encoded by 3 lamina genes:

• LMNA encodes the A-type lamina (A, A10, C, and C2).
• LMNB1 for lamin B1, LMNB2 for lamin B2 and B3

Lamines differ from the cytoplasmic intermediary filaments by a longer amino acid sequence (+ 42 amino acids) (Stuurmann et al. 1998). They also carry a nuclear localization signal. Lamines have a three-part molecular structure and a typical tertiary structure with:

• short non-helical amino-terminal head domain
• alpha-helical rod domain and
• long carboxyterminal non-alpha-helical tail

Lamin peptides have an almost complete alpha-helical shape with numerous alpha-helical protein domains, separated from non-alpha-helical links that are unchangeable in length and amino acid sequence. Both the C-terminal and the N-terminal are non-alpha-helical, the C-terminal is globular and carries a fold similar to an immunoglobulin domain. The molecular weight of the laminae polypeptides ranges from 60 to 80 kDa.

Lamin as components of the core lamina (lamina): Lamines are an essential component of the core lamina. The core lamina is a dense network of proteins that interacts with nuclear membrane proteins. The lamina is located in the cell nucleus on the inner side of the nuclear membrane. It is closely connected to the nuclear membrane via the lamina-binding membrane proteins, also in functional terms. The most important membrane proteins are LAP1 and LAP2 (for lamin associated protein), Emerin, lamin B receptor (LBR), Otefin, MAN1 and the Nesprine. In addition, lamines also occur as so-called "nuclear reticules" in the nucleoplasm. The nuclear lamina itself has an average layer thickness of 10-50 (60) nm (Liu et al. 2000) and contributes to the structural stability of the cell nucleus. Furthermore, the lamina plays a role in important processes such as DNA replication, transcription or the organisation of chromatin (Gruenbaum et al., 2003; 2005).

The molecular structure of the lamina: The lamina form class V of the 6 classes of the intermediate filament protein family. The IF proteins have molecular masses between 60 and 80 kDa and assemble into filaments with a diameter of 7-12 nm, which, among other things, make up parts of the cytoskeleton (Stewart 1993).

In humans (and mammals) there are 4 type A laminar isoforms (lamin A, C, AΔ10 and C2), which are encoded by a single gene (LMNA, 1q21) and are produced by alternative splicing. The type A laminae differ only in their C-terminal tail domains and are otherwise completely identical (Fisher et al. 1986). Type A laminae are expressed in a tissue- and development-specific manner. The C2 laminate is found exclusively in sperm. In human cells, lamin AΔ10 can be detected which differs from normal lamin A by the absence of exon 10. However, the function or the expression profile is still unknown (Machiels et al. 1996).

Type B laminae remain associated with membrane vesicles during mitosis. In humans, type B laminae are encoded by two genes, LMNB1 and LMNB2. The lamin B2 gene produces 2 splice isoforms, lamin B2 and the germline-specific lamin B3. Type B laminae are ubiquitously expressed in all cells during early embryonic development and are essential for cell survival.

The function of the laminae: The laminae play an important role during mitosis, both in dissociation and in the reconstruction of the nuclear envelope. The dissociation of the nuclear envelope is induced by the phosphorylation of a number of proteins by a cell cycle dependent p34-CDC2 kinase. These include nuclear pore complex proteins, lamina binding proteins (LAP1; LBR) and the lamina itself (Favreau et al. 1996). Phosphorylation of the lamina leads to depolymerisation of the lamina network, which results in dissolution of the lamina and finally the nuclear envelope (Spann et al. 1997). While the daughter chromosomes are separated, the lamines are still mobile. Lamines are nearly immobile after the formation of a new nuclear envelope, when they have reassembled into a dense network in the lamina (Moir et al., 2000; Lopez-Soler et al. 2001).

Another important process in which the lamina are involved is the post-mitotic reorganisation of the nucleus and the nuclear pore complexes. If the lamination content in human cells is reduced, an increased instability of the nuclear envelope as well as a conspicuous redistribution of NPCs with abnormal aggregates could be observed. This leads to premature cell death (Schirmer et al. 2004). Lamines are also involved in DNA repair processes. The lamina also plays an important role in the organization of chromatin during interphase. Direct interaction of the lamina with chromatin and with certain DNA sequences (MARs = "matrix attachment regions") and SARs (= "scaffold attachment regions") can be detected (Zhao et al. 1996). Furthermore, chromatin binds to the lamina protein lamin B1 via specific DNA regions, the so-called LADs (lamina-associated domains) (Guelen et al. 2008). Furthermore, the lamines interact indirectly with other nuclear envelope proteins via their numerous interactions and are also involved in other chromatin-associated processes. For example, through their interaction with RNA polymerase II and specific transcription factors, lamines influence the transcription of corresponding genes (Spann et al., 2002). Another transcription factor is the "Sterol Regulatory Element-Binding Protein-1" (SREBP-1) which is required for cholesterol biosynthesis in fat cells. A degeneration of adipocytes is caused by a defective binding of type A lamines to SREBP-1 (Favreau C et al. 1996). Also, cells of progeria patients who are carriers of type A lamina mutations also show morphological changes that are characteristic for apoptosis (Scaffidi et al. 2006).

Mutations in the LMNA gene lead to pathological lamines, which make the cell nucleus unstable and lead to the development of so-called laminopathies. This group of diseases, which includes muscular dystrophies, manifests itself shortly after birth and often leads to early death. These include:

• Emery-Dreifuss muscular dystrophy
• Familial partial lipodystrophy
• Limb girdle muscular dystrophy
• Dilated cardiomyopathy
• Charcot-Marie-Tooth disease
• Lethal restrictive dermopathy.
• In Hutchinson-Gilford-Progeria syndrome (Progeria infantilis) there is a deletion of 50 amino acids in prelamine A (amino acids 607-656). removes the site for the second endoproteolytic cleavage. Consequently, no mature lamin A is formed and a farnesylated mutant prelamine A (progerin) accumulates in cells.

1. Favreau C et al (1996) Cell cycle-dependent phosphorylation of nucleoporins and nuclear pore membrane protein Gp210. Biochemistry 35: 8035-8044.
2. Fisher DZ et al (1986) cDNA sequencing of nuclear lamins A and C reveals primary and secondary structural homology to intermediate filament proteins. Proc Natl Acad Sci U S A 83: 6450-6454 Gruenbaum Y et al. (2003) The nuclear lamina and its functions in the nucleus. Int Rev Cytol 226: 1-62.
3. Gruenbaum Y et al. (2005) The nuclear lamina comes of age. Nat Rev Mol Cell Biol 6: 21-31.
4. Guelen L et al (2008) Domain organization of human chromosomes revealed by mapping of nuclear lamina interactions. Nature 453: 948-951.
5. Liu Y et al (2006) DNA damage responses in progeroid syndromes arise from defective maturation of prelamin A. J Cell Sci 119: 4644-4649.
6. Lopez-Soler RI et al (2001) A role for nuclear lamins in nuclear envelope assembly. J Cell Biol 154: 61-70.
7. Machiels BM et al. (1996) An alternative splicing product of the lamin A/C gene lacks exon 10 J Biol Chem, 271: 9249-9253
8. Moir RD et al (2000) Nuclear lamins A and B1: different pathways of assembly during nuclear envelope formation in living cells. J Cell Biol 151: 1155-1168.
9. Scaffidi P et al. (2006) Lamin A-dependent nuclear defects in human aging. Science 312: 1059-1063.
10. Schirmer EC et al (2004) The stability of the nuclear lamina polymer changes with the composition of lamin subtypes according to their individual binding strengths. J Biol Chem 279: 42811-42817.
11. Spann TP et al (2002) Alteration of nuclear lamin organization inhibits RNA polymerase II-dependent transcription. J Cell Biol 156: 603-608.
12. Spann TP et al (1997) Disruption of nuclear lamin organization alters the distribution of replication factors and inhibits DNA synthesis. J Cell Biol 136: 1201-1212.
13. Stewart M (1993) Intermediate filament structure and assembly. Curr Opin Cell Biol 5: 3-11.
14. Stuurman N et al (1998) Nuclear lamins: their structure, assembly, and interactions. J Struct Biol 122: 42-66.
15. Zhao K et al (1996) Binding of matrix attachment regions to nuclear lamin is mediated by the rod domain and depends on the lamin polymerization state. FEBS Lett 380: 161-164.