The nuclear envelope (NE), a highly regulated double-membrane barrier, is a defining feature of the eukaryotic nucleus. Long established for its role in separating the nucleus from the cytoplasm, thereby protectively enclosing the nuclear genome within, and providing structural integrity to the nucleus, there is mounting evidence indicating its involvement and that of its integral proteins in various cell functions, including the cell cycle, DNA replication, apoptosis, gene transcription and regulation, and chromatin organization. Thus, the NE, which comprises of four key components, viz. (i) the nuclear pore complex (NPC) (ii) the outer nuclear membrane (ONM) (iii) the inner nuclear membrane (INM) and (iv) the nuclear lamina (Fig.1), is beginning to be acknowledged as a key upstream component involved in several major cellular processes. (Hetzer 2010)
Figure .1: Topology of the Nuclear Envelope (Hetzer 2010)
Of special intrigue in this project are the nuclear lamina, its associated INM protein network and their global genomic effect. Dysregulation and defects in any of these components triggers a nuclear/cellular pandemonium involving nuclear disruption, DNA damage, anomalous expression and misexpression of essential genes, breakdown of homeostasis and an ultimate systemic failure. These events have been well characterized in the many laminopathies and progeroid disorders, which provide an indication of the role of lamins as master regulators of gene expression and chromatin organization.
1.1 The Peripheral Lamina
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The presence of the nuclear lamina was first reported in 1966 (Fawcett 1966) (Fig.1.1), observed as a complex proteinaceous meshwork lying below the INM and closely associated with the INM proteins, while being attached via specific factors to the peripheral heterochromatin. The lamina itself is made up of only lamin protofilaments, constituting of merely a maximum of four lamins, viz. lamins A, C, B1 and B2.
Figure 1.2: Transition Electron Micrograph Fibrous Nuclear Lamina located between the Peripheral Heterochromatin and INM (Fawcett 1966)
Cell cycle mitosis
Many associated proteins of the lamina are integral proteins of the INM. These proteins localize by binding directly or indirectly to lamin filaments. The network thus formed at the INM is known as the peripheral lamina (Wilson and Foisner 2010). Lamins are important structurally and as scaffolds for many other proteins and complexes in the nucleus. The resultant lamin-INM protein interactions effect several nuclear, cellular and extracellular events as well as signalling cascades, as is depicted in Fig. 1.3. Documented in Table 1.I are some important lamin-binding INM proteins and their known functions.
Table 1.II: Important Lamin-binding INM Proteins and their Functions *
LEM domain proteins
LAP2 (Lamin Associated Polypeptide-2)
binds lamin B specifically
directly interacts with transcription regulators and epigenetic modifiers
contributes to transcriptional repression
Interact via the LEM motif with the DNA cross-linking protein BAF (Barrier-to-Autointegration Factor) therefore leads to chromatin tethering, hence have a role in the formation and epigenetic control of heterochromatin
localizes to the nuclear interior, and specifically binds A-type lamins
Lap2a-lamin complexes are involved in Rb regulation including repression of E2F/Rb target genes.
binds to all lamins
has a large number of interactors including other INM proteins, BAF, structural proteins, and proteins involved in signalling, transcription and mRNA splicing
important component of various multiprotein complexes which include other nuclear architecture factors and chromatin and transcription regulators
is dispensable for cell survival and normal development
directly binds and inhibits R-Smads
functions as a major regulator of BMP- and TGFÎ²-signalling in early vertebrate development
important in early development for chromosome segregation and cell division that are essential for cell survival
Lamin-B Receptor (LBR)
is a sterol reductase that binds B-type lamins
required for nuclei to change shape and reorganize chromatin in differentiating neutrophils
proposed to associate with heterochromatic under-acetylated chromatin through binding to heterochromatin protein 1 (HP1)
Interacts with B-type lamins and HP1 in conjunction with core histones
SUN (Sad1p/UNC-84) domain proteins
interact as dimers with KASH domain proteins (nesprins) of the ONM in the NE lumen which in turn interact with actin, microtubules, centrosomes, plectins and IFs of the cytoplasm
Always on Time
Marked to Standard
mediate several nuclear features including NPC positioning, controlling NE architecture, force-transfer across the NE and stiffness in the cytoskeleton
Important members of the LINC (Linkers of the Nucleoskeleton and the Cytoskeleton) complex (Starr & Fridolfsson 2010)
* (Broers et al. 2006; Vlcek & Foisner 2007; Wilson & Foisner 2010; Hetzer 2010)
Nuclear lamins are characterized as Type V intermediate filament (IF) proteins, types I-IV constituting the cytoskeleton (Aebi et al. 1986). A further classification into two major groups as A-type and B-type lamins is based on their sequence homology, expression patterns, structural features and biochemical and dynamic properties (Broers et al. 2006; Dechat et al. 2010).
Fig 1.4: (Howard J Worman et al. 2009)
In its entirety, lamins comprise of seven isoforms arising from three genes. A single LMNA gene encodes for four different alternatively spliced products, viz. predominantly lamin A and lamin C, and tissue- and developmental-stage specific AÎ”10 and C2 (Lin & Worman 1993). On the other hand, lamin B1 is a product of the LMNB1 gene while lamin B2, and to a lesser extent B3, are encoded by the LMNB2 gene. Table 1.III and Fig.1.4 summarize the human lamin gene sequences:
Lamin polypeptide chains, as in the case of cytoplasmic IF proteins, possess a typical tripartite structure comprising of a highly conserved Î±-helical central rod flanked by a globular amino-terminal head and a lesser conserved globular carboxy-terminal tail. The central rod is comprised of four domains viz 1A, 1B, 2A and 2B, arranged as heptad repeats, alternated with linker regions L1, L2 and L12 (Fig. 1.5). Three important features characteristic of only the lamin proteins are present on the C-terminal tail: (i) a conserved structural motif: the immunoglobulin (Ig) fold; (ii) the nuclear localization signal (NLS): to aid in lamin transport across the the nuclear membrane and, most importantly (iii) the CAAX box forming the terminal end: present on most A- and B-type lamins (except lamin C) crucial for post-translational modification and subsequent targeting to the INM.
Figure 1.5: Schematic Characteristics of Lamin Proteins (Broers et al. 2006)
Two parallel and in-register lamin protein chains dimerize forming a coiled coil structure through the association of their rod domains which is the basic structural unit of the nuclear lamina. Lamin dimers assemble in a head-to-tail fashion as polarized arrays that interact in an antiparallel fashion to form apolar tetrameric protofilaments that interact laterally to form 10nm diameter filaments and higher-order structures (Heitlinger et al. 1991).
Amongst the various lamin isoforms, B-type lamins are ubiquitously present in all metazoan cells while A-type lamin expression is developmentally regulated. Most significantly, A-type lamins are absent in undifferentiated cells, cells of hematopoietic lineage as well as the early embryo. In many organs, lamins A/C are expressed only after birth whereas B-type lamins are perpetually expressed throughout the metazoan cell's lifespan (Constantinescu et al. 2006). These phenomena have been experimentally demonstrated in LMNA knockout (LMNA-/-) and LMNB1 mutant (LMNBÎ”/Î”) mice. In case of the former, the embryo develops normally, but postnatally, the organism exhibits a drastic phenotype including growth retardation and muscular dystrophy (Sullivan et al. 1999). In the latter, mutations in any B-type lamin are lethal and result in death at birth due to defects during embryogenesis (Vergnes et al. 2004). As such, B-type lamins are essential for cell viability and development, whereas A-type lamins arose later in evolution and are nonessential.
The Lamin class of proteins are seen to exist only in Metazoans, evolutionarily observed earliest in Nematodes. The current belief is that unicellular organisms and plant nuclei lack lamins or its homologues, as their presence in these systems have not yet been demonstrated. However, in most inverterbrates only a single B-type lamin is seen to occur, with the exception of Drosophila, which has a lamin each of A-type (Lamin C) and B-type (Dm0). Amongst vertebrates, most commonly one A-type and two B-type lamins is what is observed except for certain amphibians, that are known to have one A-type and three B-type (B1, B2 and B3) lamins and mammals, which have four lamins: A,C, B1 and B2.
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(Erber et al. 1999)
1.3 Post Translational Modification Processes
As previously mentioned, the evolutionarily conserved CAAX box at the prelamin carboxy-terminal tail directs their post-translational modification via protein prenylation (Beck et al. 1988). The 'C' denotes a Cysteine residue, 'AA' denotes two aliphatic amino residues and 'X' is any C-terminal amino residue, most commonly Methionine, Serine, Alanine, Cysteine and Glutamine. In lamins, the CAAX box is inherently the target of the 15-carbon farnesyl isoprenoid, a product of the mevalonate pathway, which is attached to the Cysteine residue. This lipid modification is essential for the correct attachment of the lamins to the INM and therefore proper placement of the nuclear lamina (Nigg et al. 1992; Hennekes & Nigg 1994)
The farnesylation modification is an enzymatic process that generally involves three key steps as depicted in Fig.--:
(Dechat et al. 2010)
Shortly after pre-lamin synthesis, (i) a farnesyl group is attached to the Cysteine residue of the terminal CAAX box via a farnesyltransferase (FT) to the translated pre-lamin sequence. This is followed by (ii) a proteolytic step that involves the cleavage of the '-AAX' tripeptide, which leaves the Cysteine residue carboxylated. Of our interest here is the AAX endopeptidase activity containing zinc metalloprotease enzymes, viz. the FACE (farnesylated-proteins converting enzymes) family, well characterized in mammals as Face1 and Face2, more commonly known as Zmpste24 (zinc metalloprotease like Ste24) and Rce1 (Ras converting enzyme 1), respectively. The current view is that this step is catalyzed by Zmste24 for A-type lamins and by Rce1 in case of B-type lamins. As will be discussed ahead, mutations in these enzymes have major repercussions on proper lamin maturation ultimately leading to a host of laminopathies. Finally, this Cysteine residue is carboxymethylated by the isoprenyl-Cysteine methyltransferase (ICMT), and in most cases, this results in a mature lamin protein (Rusiñol & Sinensky 2006, Gao et al. 2009)
The process outlined above is true for insoluble proteins permanently farnesylated and carboxymethylated. In case of lamins, this corresponds to the B-type lamin wherein the retained hydrophobic group function as the anchor to the INM. However, A-type lamins, which are essentially soluble lamins that sometimes are seen to be present within the nucleoplasm independent of the lamina (Hozák et al. 1995, Dechat et al. 2011), necessitates a further farnesyl-group removal step for their complete maturation. This secondary endoproteolysis occurs 15 amino acid residues upstream to the carboxymethylated Cysteine, at the highly conserved RSYLLG hexapeptide sequence, and involves Zmpste24 (Face1) specifically for one or both the reactions. The resultant
Knockout studies in mice have fu
It is important to note that the maturation of lamin C from pre-lamin A
Figure : Post-translational processing of Lamins A and B
Recent years have revealed intriguing new aspects of the mechanical and gene regulatory functions of lamin complexes and their potential relevance for laminopathic diseases.
(Broers et al. 2006)
(Capell & Collins 2006)(Coutinho et al. 2009)