BIOLOGICAL OVERVIEW

Nuclear lamins belong to the intermediate filament (IF) superfamily of proteins that includes type I and II IFs called keratins (a component of wool fibers), type III IF (such a vimentin, desmin and peripherin), type IV IF (expressed in axons, dendrites and perikarya), and type V IF, proteins making up nuclear lamina (Fuchs, 1994). Drosophila Lamin is one of two nuclear lamins. Lamin is expressed constitutively, in contrast to the second Drosophila lamin, Lamin C, which is developmentally regulated (Riemer, 1995). Lamins are the major structural proteins of the nuclear lamina, a structure that lines the nucleoplasmic surface of the inner nuclear membrane in higher eukaryotic cells. The nuclear lamina is composed of a meshwork of 10 nm filaments that are thought to provide a skeletal support for the nuclear envelope and to mediate the attachment of the nuclear envelope to interphase chromatin. Additional functions of the nuclear lamina may include the proper organization and anchoring of nuclear pore complexes. During mitosis the lamins also play a crucial role in the disassembly and reassembly of the nuclear envelope (Lenz-Bohme, 1997 and references).

Before describing the biological properties of Drosophila Lamin, the general properties of the IF superfamily members will be described. IF proteins are all predicted to share a common secondary structure. All IF proteins have a central alpha-helical domain, the rod, which is flanked by nonhelical head (amino-end) and tail (carboxy-end) domains. The rods of two polypeptide chains intertwine in a coiled-coil fashion. Throughout the alpha-helical sequences are repeats of hydrophobic amino acids, such that the first and fourth repeats of every seven residues are frequently apolar. This provides a hydrophobic seal on the helical surface, enabling the coiling between two IF polypeptides. The IF alpha-helical rod is subdivided by three short nonhelical linker segments, which often contain proline or multiple glycine residues (Fuchs, 1994).

For all IFs, the first step in assembly is the formation of parallel, in-register dimers. Upon assembly, lamin dimers align in a head-to-tail fashion to form linear polymers. Most IF proteins can form functional homodimers. Each 10-nm IF filament is composed of smaller protofibrils. IFs appear to have approximately four protofibrils per unit width. It is thought that the conserved amino ends of IF proteins play an important role in assembly of the 10-Nm filament structure. One role of IF tails may be to control lateral associations at the protofilament and protofibril level, thereby influencing filament diameter. In addition, the IF head might promote lateral associations of protofilaments and protofibrils. It is known that headless lamins cannot form a linear array of dimers typical of tail-less and wild-type lamins, suggesting that the lamin head might function in tetramer elongation. Phosphorylation is known to negatively regulate IF assembly. It is thought that the archetypal IF gene had a laminlike structure (Fuchs, 1994 and references).

Lamin Dm0, the precursor form of Lamin, has an apparent molecular mass of 76 kDa and is rapidly processed proteolytically in the cytoplasm into a form migrating at 74 kDa (Lamin Dm1). Lamin Dm1 is imported into the nucleus, where about 50% is posttranslationally modified into a slower migrating form (75 kD) called Lamin Dm2. In vivo pulse-chase studies indicated that lamins Dm1 and Dm2 are in equilibrium. Treatment of lamins Dm1 and Dm2 with phosphatase results in a single form that comigrates with Lamin Dm1 (Smith, 1987 and Smith, 1989). Lamins Dm1 and Dm2 are present as a random mixture of homo- and heterodimers. It is thought that serine 25 is the Lamin Dm2-specific phosphorylation site (Stuurman, 1995).

Lamin is known to interact directly with highly conserved sequences of DNA. Lamin binds with high affinity to scaffold/matrix-associated regions (M/SARs). These DNA sequences are held responsible for mediating the interaction between the nuclear matrix and chromatin. M/SARs are several hundred base pairs long and contain stretches of AT-rich sequences that are likely to form an open chromatin configuration. Indeed, the binding of M/SARs to lamin polymers involves single-stranded regions. In addition, this binding is saturable and requires the minor groove. Lamin polymers also bind to Drosophila centromeric and telomeric sequences. The polymerized alpha-helical rod domain of Lamin, on its own, provides for specific binding to the fushi tarazu M/SAR (Zhao, 1996). The ftz M/SAR functions as an autonomously replicating sequence (ARS) in the budding yeast S. cerevisiae. This M/SAR is found in a 2.57 kb ftz upstream regulatory element. A 189 base pair minimal fragment has ARS function. However, based on growth rates and mitotic stability, its activity is lower than that of the entire SAR. The addition of flanking sequences, including as little as 100 bp of AT-rich DNA to the left of the minimal sequence, can enhance the replicative ability of the ARS. These results implicate lamins in initiation of DNA replication (Amati, 1990 a and b)

Several proteins are associated with the nuclear lamina, and specifically with Lamin proteins. The interaction of lamins with the inner nuclear membrane may be supported by integral membrane proteins, e.g., the putative lamin receptor p54 (Bailer, 1991) or LAPs, the lamina-associate proteins (Foisner, 1993).

One protein associated with Drosophila Lamin is Otefin. Otefin is the corruption and transliteration of the Hebrew word "otef," meaning envelope. Otefin is a peripheral protein of the inner nuclear membrane in Drosophila. During nuclear assembly in vitro, it is required for the attachment of membrane vesicles to chromatin. Otefin colocalizes with Lamin derivatives in situ and presumably in vivo and is present in all somatic cells examined during the different stages of Drosophila development. Otefin is a phosphoprotein in vivo and is a substrate for in vitro phosphorylation by cdc2 kinase and cyclic AMP-dependent protein kinase. It is suggested that Otefin plays a role in the assembly of the Drosophila nuclear envelope (Ashery-Padan, 1997b).

The fs(1)Ya protein (Ya stands for "young arrest") is an essential, maternally encoded, nuclear lamina protein that is under both developmental and cell cycle control. A strong Ya mutation results in early arrest of embryos. Ya mutant embryos arrest with abnormal nuclear envelopes prior to the first mitotic division. Ya unfertilized eggs contain nuclei of different sizes and condensation states, apparently due to abnormal fusion of the meiotic products immediately after meiosis. Lamin is localized at the periphery of the uncondensed nuclei in these eggs. These results suggest that Ya function is required during and after egg maturation to facilitate proper chromatin condensation, rather than to allow a lamin-containing nuclear envelope to form. Ya might bind to chromatin and organize the chromatin structure in early embryos in a way that permits DNA replication. Whether Ya functions in conjunction with lamin is unknown (Liu, 1995).

A Drosophila Lamin mutant shows a severe phenotype: this includes retardation in development, reduced viability, sterility, and impaired locomotion. Mutant adult flies die within 2 weeks after eclosion. Late stages of oogenesis are rarely detected in mutant ovaries, and the egg chambers present show an abnormal morphology. In heads from homozygous mutant flies, Lamin is significantly decreased in nuclei of the densely packed cell bodies of the central nervous system. Reduced Lamin expression causes an enrichment of nuclear pore complexes in cytoplasmic annulate lamellae and in nuclear envelope clusters. Annulate lamellae are stacked sheets of membranes in the cytoplasm that contain pore complexes in high densities and are often continuous with rough endoplasmic reticulum in several cells, particularly the densely packed somata of the central nervous system. Defective nuclear envelopes are also observed. The lack of full lethality at early developmental stages may be due to maternal transmission from heterozygous mothers. Indeed, Lamin protein is highly enriched inside the oocyte nucleus, which may serve as a storage compartment for lamin required during the early nuclear divisions in the embryo. These data constitute the first genetic proof that lamins are essential for the structural organization of the cell nucleus (Lenz-Bohme, 1997).

GENE STRUCTURE

The Drosophila Lamin gene is developmentally regulated, giving rise to a 2.8 kb maternal transcript and a 3.0 kb zygotic transcript. The different transcripts are generated by utilizing different polyadenylation sites. None of the putative lamin polyadenylation signals contains the consensus AAUAA sequence. The choice between the different polyadenylation signals might depend on maternal fators that more efficiently recognize the polyadenylation signal of the 2.8 kb transcript (Osman, 1990 and Gruenbaum, 1988)

Exons - 4

Bases in 3' UTR - 710 (maternal) and 921 (zygotic)

PROTEIN STRUCTURE

Amino Acids - 621

Structural Domains

Highly specific features of lamins include a nuclear localization signal, a C-terminal CaaX sequence (where C=cysteine; a=aliphatic amino acid; X=any amino acid) and characteristic phosphorylation sites in the N-terminal head and C-terminal tail domains. The nuclear localization signal is responsible for rapid transport of lamins into the nucleus, thus preventing cytoplasmic assembly. Modification by isoprenylation and carboxymethylation at the CaaX motif targets lamins to the inner nuclear membrane (Lenz-Bohme, 1997 and references).

date revised: 16 July 97  

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