Overexpression of the lamina proteins Lamin and Kugelkern induces specific ultrastructural alterations in the morphology of the nuclear envelope of intestinal stem cells and enterocytes

The nuclear envelope has a stereotypic morphology consisting of a flat double layer of the inner and outer nuclear membrane, with interspersed nuclear pores. Underlying and tightly linked to the inner nuclear membrane is the nuclear lamina, a proteinous layer of intermediate filament proteins and associated proteins. Physiological, experimental or pathological alterations in the constitution of the lamina lead to changes in nuclear morphology, such as blebs and lobulations. It has so far remained unclear whether the morphological changes depend on the differentiation state and the specific lamina protein. Here we analysed the ultrastructural morphology of the nuclear envelope in intestinal stem cells and differentiated enterocytes in adult Drosophila flies, in which the proteins Lam, Kugelkern or a farnesylated variant of LamC were overexpressed. Surprisingly, we detected distinct morphological features specific for the respective protein. Lam induced envelopes with multiple layers of membrane and lamina, surrounding the whole nucleus whereas farnesylated LamC induced the formation of a thick fibrillary lamina. In contrast, Kugelkern induced single-layered and double-layered intranuclear membrane structures, which are likely be derived from infoldings of the inner nuclear membrane or of the double layer of the envelope.     

Trypanosoma cruzi: Identification of DNA targets of the nuclear periphery coiled-coil protein TcNUP-1

The nuclear lamina is a structure that lines the inner nuclear membrane. In metazoans, lamins are the primary structural components of the nuclear lamina and are involved in several processes. Eukaryotes that lack lamins have distinct proteins with homologous functions. Some years ago, a coiled-coil protein in Trypanosoma brucei, NUP-1, was identified as the major filamentous component of its nuclear lamina. However, its precise role has not been determined. We characterized a homologous protein in Trypanosoma cruzi, TcNUP-1, and identified its in vivo DNA binding sites using a chromatin immunoprecipitation assay. We demonstrate for the first time that TcNUP-1 associates with chromosomal regions containing large non-tandem arrays of genes encoding surface proteins. We therefore suggest that TcNUP-1 is a structural protein that plays an essential role in nuclear organization by anchoring T. cruzi chromosomes to the nuclear envelope.     

Involvement of the lamin rod domain in heterotypic lamin interactions important for nuclear organization

The nuclear lamina is a meshwork of intermediate-type filament proteins (lamins) that lines the inner nuclear membrane. The lamina is proposed to be an important determinant of nuclear structure, but there has been little direct testing of this idea. To investigate lamina functions, we have characterized a novel lamin B1 mutant lacking the middle approximately 4/5 of its alpha-helical rod domain. Though retaining only 10 heptads of the rod, this mutant assembles into intermediate filament-like structures in vitro. When expressed in cultured cells, it concentrates in patches at the nuclear envelope. Concurrently, endogenous lamins shift from a uniform to a patchy distribution and lose their complete colocalization, and nuclei become highly lobulated. In vitro binding studies suggest that the internal rod region is important for heterotypic associations of lamin B1, which in turn are required for proper organization of the lamina. Accompanying the changes in lamina structure induced by expression of the mutant, nuclear pore complexes and integral membrane proteins of the inner membrane cluster, principally at the patches of endogenous lamins. Considered together, these data indicate that lamins play a major role in organizing other proteins in the nuclear envelope and in determining nuclear shape.     

Nuclear lamina at the crossroads of the cytoplasm and nucleus

The nuclear lamina is a protein meshwork that lines the nuclear envelope in metazoan cells. It is composed largely of a polymeric assembly of lamins, which comprise a distinct sequence homology class of the intermediate filament protein family. On the basis of its structural properties, the lamina originally was proposed to provide scaffolding for the nuclear envelope and to promote anchoring of chromatin and nuclear pore complexes at the nuclear surface. This viewpoint has expanded greatly during the past 25 years, with a host of surprising new insights on lamina structure, molecular composition and functional attributes. It has been established that the self-assembly properties of lamins are very similar to those of cytoplasmic intermediate filament proteins, and that the lamin polymer is physically associated with components of the cytoplasmic cytoskeleton and with a multitude of chromatin and inner nuclear membrane proteins. Cumulative evidence points to an important role for the lamina in regulating signaling and gene activity, and in mechanically coupling the cytoplasmic cytoskeleton to the nucleus. The significance of the lamina has been vaulted to the forefront by the discovery that mutations in lamins and lamina-associated polypeptides lead to an array of human diseases. A key future challenge is to understand how the lamina integrates pathways for mechanics and signaling at the molecular level. Understanding the structure of the lamina from the atomic to supramolecular levels will be essential for achieving this goal.     

Breaking and making of the nuclear envelope

During mitosis, a single nucleus gives rise to two nuclei that are identical to the parent nucleus. Mitosis consists of a continuous sequence of events that must be carried out once and only once. Two such important events are the disassembly of the nuclear envelope (NE) during the first stages of mitosis, and its accurate reassembly during the last stages of mitosis. NE breakdown (NEBD) is initiated when maturation-promoting factor (MPF) enters the nucleus and starts phosphorylating nuclear pore complexes (NPCs) and nuclear lamina proteins, followed by NPC and lamina breakdown. Nuclear reassembly starts when nuclear membranes assemble onto the chromatin. This article focuses on the different models of NEBD and reassembly with emphasis on recent data.     

Herpes simplex virus infection induces phosphorylation and delocalization of emerin, a key inner nuclear membrane protein

The inner nuclear membrane (INM) contains specialized membrane proteins that selectively interact with nuclear components including the lamina, chromatin, and DNA. Alterations in the organization of and interactions with INM and lamina components are likely to play important roles in herpesvirus replication and, in particular, exit from the nucleus. Emerin, a member of the LEM domain class of INM proteins, binds a number of nuclear components including lamins, the DNA-bridging protein BAF, and F-actin and is thought to be involved in maintaining nuclear integrity. Here we report that emerin is quantitatively modified during herpes simplex virus (HSV) infection. Modification begins early in infection, involves multiple steps, and is reversed by phosphatase treatment. Emerin phosphorylation during infection involves one or more cellular kinases but can also be influenced by the US3 viral kinase, a protein whose function is known to be involved in HSV nuclear egress. The results from biochemical extraction analyses and from immunofluorescence of the detergent-resistant population demonstrate that emerin association with the INM significantly reduced during infection. We propose that the induction of emerin phosphorylation in infected cells may be involved in nuclear egress and uncoupling interactions with targets such as the lamina, chromatin, or cytoskeletal components.     

Pathway of incorporation of microinjected lamin A into the nuclear envelope

When microinjected into the cytoplasm of 3T3 cells, biotinylated human lamin A rapidly enters the nucleus and gradually becomes incorporated into the nuclear lamina region as determined by immunofluorescence. The incorporation of the microinjected material takes several hours and progresses through a series of morphologically identifiable stages. Within minutes after microinjection, lamin A is found in spots distributed throughout the nucleus, except in nucleolar regions. Over a time course of up to 6 h, these spots appear to decrease in size and number as the biotinylated lamin A becomes associated with the endogenous nuclear lamina. Eventually, the typical nuclear rim staining pattern normally revealed by immunofluorescence with nuclear lamin antibodies is seen with antibiotin. This latter rim staining property is passed on to daughter cells following mitosis. These results indicate that the microinjected biotinylated nuclear lamin A retains those properties required for its integration into the lamina, as well as those necessary for the disassembly and subsequent reassembly of the nuclear lamina during cell division. The initial rapid accumulation into foci and the subsequent slower incorporation into the nuclear lamina appear to be analogous to the stages of incorporation following the microinjection of cytoskeletal intermediate filament proteins such as vimentin and keratin (Vikstrom, K., G. G. Borisy, and R. D. Goldman. 1989. Proc. Natl. Acad. Sci. USA. 86:549-553; Miller, R. K., K. Vikstrom, and R. D. Goldman. 1991. J. Cell Biol. 113:843-855). Foci are also observed in some uninjected cells using nuclear lamin antibodies, indicating that these features are a genuine component of nuclear substructure. Evidence is presented that shows the appearance of these nuclear structures is cell cycle dependent.     

Interactions among Drosophila Nuclear Envelope Proteins Lamin, Otefin, and YA

The nuclear envelope plays many roles, including organizing nuclear structure and regulating nuclear events. Molecular associations of nuclear envelope proteins may contribute to the implementation of these functions. Lamin, otefin, and YA are the three Drosophila nuclear envelope proteins known in early embryos. We used the yeast two-hybrid system to explore the interactions between pairs of these proteins. The ubiquitous major lamina protein, lamin Dm, interacts with both otefin, a peripheral protein of the inner nuclear membrane, and YA, an essential, developmentally regulated protein of the nuclear lamina. In agreement with this interaction, lamin and otefin can be coimmunoprecipitated from the vesicle fraction of Drosophila embryos and colocalize in nuclear envelopes of Drosophila larval salivary gland nuclei. The two-hybrid system was further used to map the domains of interaction among lamin, otefin, and YA. Lamin’s rod domain interacts with the complete otefin protein, with otefin’s hydrophilic NH₂-terminal domain, and with two different fragments derived from this domain. Analogous probing of the interaction between lamin and YA showed that the lamin rod and tail plus part of its head domain are needed for interaction with full-length YA in the two-hybrid system. YA’s COOH-terminal region is necessary and sufficient for interaction with lamin. Our results suggest that interactions with lamin might mediate or stabilize the localization of otefin and YA in the nuclear lamina. They also suggest that the need for both otefin and lamin in mediating association of vesicles with chromatin might reflect the function of a protein complex that includes these two proteins.     

Integral membrane proteins specific to the inner nuclear membrane and associated with the nuclear lamina

We obtained a monoclonal antibody (RL13) that identifies three integral membrane proteins specific to the nuclear envelope of rat liver, a major 75-kD polypeptide and two more minor components of 68 and 55 kD. Immunogold labeling of isolated nuclear envelopes demonstrates that these antigens are localized specifically to the inner nuclear membrane, and that the RL13 epitope occurs on the inner membrane's nucleoplasmic surface where the nuclear lamina is found. When nuclear envelopes are extracted with solutions containing nonionic detergent and high salt to solubilize nuclear membranes and pore complexes, most of these integral proteins remain associated with the insoluble lamina. Since the polypeptides recognized by RL13 are relatively abundant, they may function as lamina attachment sites in the inner nuclear membrane. Major cross-reacting antigens are found by immunoblotting and immunofluorescence microscopy in all rat cells examined. Therefore, these integral proteins are biochemical markers for the inner nuclear membrane and will be useful models for studying nuclear membrane biogenesis.     

Nonlamin components of the lamina: a paucity of proteins.

Current models of nuclear organization propose that nuclear functions are modulated in part by reversible tethering of chromatin loops to structural elements of the nucleoplasm and the nuclear envelope. Lamins are the best-characterized proteins of the lamina portion of the nuclear envelope and are involved in binding chromatin to the inner nuclear membrane. However, they are not a universal feature of eukaryotic nuclei and do not account fully for the putative functions of the lamina in all organisms. It is possible that nonlamin components of the lamina may substitute for lamins in organisms from which they are absent and modify the properties of lamins during development and the cell cycle. We review the properties of the relatively small number of such components that have been reported, including the young arrest (fs(1)Ya) protein of Drosophila, statin, circumferin, and the MAN antigens. The experimental evidence indicates they are a diverse group of proteins, and that at least some have the potential to modulate the interactions of chromatin, lamins, and the nuclear membranes.     

Disruption of Nuclear Lamin Organization Alters the Distribution of Replication Factors and Inhibits DNA Synthesis

The nuclear lamina is a fibrous structure that lies at the interface between the nuclear envelope and the nucleoplasm. The major proteins comprising the lamina, the nuclear lamins, are also found in foci in the nucleoplasm, distinct from the peripheral lamina. The nuclear lamins have been associated with a number of processes in the nucleus, including DNA replication. To further characterize the specific role of lamins in DNA replication, we have used a truncated human lamin as a dominant negative mutant to perturb lamin organization. This protein disrupts the lamin organization of nuclei when microinjected into mammalian cells and also disrupts the lamin organization of in vitro assembled nuclei when added to Xenopus laevis interphase egg extracts. In both cases, the lamina appears to be completely absent, and instead the endogenous lamins and the mutant lamin protein are found in nucleoplasmic aggregates. Coincident with the disruption of lamin organization, there is a dramatic reduction in DNA replication. As a consequence of this disruption, the distributions of PCNA and the large subunit of the RFC complex, proteins required for the elongation phase of DNA replication, are altered such that they are found within the intranucleoplasmic lamin aggregates. In contrast, the distribution of XMCM3, XORC2, and DNA polymerase α, proteins required for the initiation stage of DNA replication, remains unaltered. The data presented demonstrate that the nuclear lamins may be required for the elongation phase of DNA replication.     

Nuclear envelope proteins from Spisula solidissima germinal vesicles

Biochemical characterization of the nuclear pore complex requires quantities of highly enriched nuclear pore complex material which could not be obtained with available procedures. We have developed a technique for the mass isolation of nuclear envelopes from germinal vesicles of Spisula solidissima oocytes. The nuclear pore complex is intact after isolation as judged ultrastructurally. The nuclear envelope and the pore complex fibrous lamina fraction are highly purified with respect to nuclear and cytoplasmic protein contaminants. The fibrous lamina pore complex (FLPC) as presently isolated consists of about eight major proteins, three of which are phosphorylated. Comparison of the FLPC of clams with that of rat reveals three proteins of similar molecular weights, which may be pore complex-specific proteins. The clam nuclear envelope has only one protein (67000) in the molecular weight range which is comparable to the three lamina of rat nuclei. The solubility, intermolecular cross-linking and in vitro phosphorylation of this protein resemble that of one of the lamina of rat nuclei. The other lamina of the rat nuclear envelope are not essential proteins of the pore complex because they are not present in the clam FLPC preparation. They also seem non-essential for the maintenance of the fibrous lamina.     

Review: nuclear lamins--structural proteins with fundamental functions

The nuclear lamina is located between the inner nuclear membrane and the peripheral chromatin. It is composed of both peripheral and integral membrane proteins, including lamins and lamina-associated proteins. Lamins can interact with one another, with lamina-associated proteins, with nuclear scaffold proteins, and with chromatin. Likewise, most of the lamina-associated proteins are likely to interact directly with chromatin. The nuclear lamina is required for proper cell cycle regulation, chromatin organization, DNA replication, cell differentiation, and apoptosis. Mutations in proteins of the nuclear lamina can disrupt these activities and cause genetic diseases. The structure and assembly of the nuclear lamina proteins and their roles in chromatin organization and cell cycle regulation were recently reviewed. In this review, we discuss the roles of the nuclear lamina in DNA replication and apoptosis and analyze how mutations in nuclear lamina proteins might cause genetic diseases.     

In vitro disassembly of the nuclear lamina and M phase-specific phosphorylation of lamins by cdc2 kinase

The nuclear lamina is an intermediate filament-type network underlying the inner nuclear membrane. Phosphorylation of lamin proteins is believed to cause lamina disassembly during meiotic and mitotic M phase, but the M phase-specific lamin kinase has not been identified. Here we show that the cdc2 kinase, a major element implicated in controlling the eukaryotic cell cycle, phosphorylates chicken B-type lamins in vitro on sites that are specifically phosphorylated during M phase in vivo. Concomitantly, cdc2 kinase is capable of inducing lamina depolymerization upon incubation with isolated nuclei. One of the target sites of cdc2 kinase is identified as a motif (SPTR) conserved in the N-terminal domain of all lamin proteins. These results lead us to propose that mitotic disassembly of the nuclear lamina results from direct phosphorylation of lamins by cdc2 kinase.     

Purification of the vertebrate nuclear pore complex by biochemical criteria

The nuclear pore is a large and complex biological machine, mediating all signal-directed transport between the nucleus and the cytoplasm. The vertebrate pore has a mass of ∼120 million daltons or 30 times the size of a ribosome. The large size of the pore, coupled to its tight integration in the nuclear lamina, has hampered the isolation of pore complexes from vertebrate sources. We have now developed a strategy for the purification of nuclear pores from in vitro assembled annulate lamellae (AL), a cytoplasmic mimic of the nuclear envelope that lacks a lamina, nuclear matrix, and chromatin-associated proteins. We find that purified pore complexes from annulate lamellae contain every nuclear pore protein tested. In addition, immunoblotting reveals the presence of soluble transport receptors and factors known to play important roles in the transport of macromolecules through the pore. While transport factors such as Ran and NTF2 show only transient interaction with the pores, a number of soluble transport receptors, including importin β, show a tight association with the purified pores. In summary, we report that we have purified the vertebrate pore by biochemical criteria; silver staining reveals ∼40–50 distinct protein bands.     

Chromatin organization in relation to the nuclear periphery

In the limited space of the nucleus, chromatin is organized in a dynamic and non-random manner. Three ways of chromatin organization are compaction, formation of loops and localization within the nucleus. To study chromatin localization it is most convenient to use the nuclear envelope as a fixed viewpoint. Peripheral chromatin has both been described as silent chromatin, interacting with the nuclear lamina, and active chromatin, interacting with nuclear pore proteins. Current data indicate that the nuclear envelope is a reader as well as a writer of chromatin state, and that its influence is not limited to the nuclear periphery.     

Composition, synthesis, and assembly of the embryonic chick retinal basal lamina

To study the biology of basal laminae in the developing nervous system the protein composition of the embryonic retinal basal lamina was investigated, the site of synthesis of its proteins in the eye was determined, and basal lamina assembly was studied in vivo in two assay systems. Laminin, nidogen, agrin, collagen IV, and XVIII are major constituents of the retinal basal lamina. However, only agrin is synthesized by the retina, whereas the other matrix constituents originate from cells of the ciliary body, the lens, or the optic disc. The synthesis from extraretinal tissues infers that the retinal basal lamina proteins must be shed from their tissues of origin into the vitreous body and from there bind to receptor proteins provided by the retinal neuroepithelium. The fact that all proteins typical for the retinal basal lamina are abundant in the vitreous body and a new basal lamina is only formed when the vitreous body was directly adjacent to the retina is consistent with the contention of the vitreous body having a function in retinal basal lamina formation. Basal lamina assembly was also studied after disrupting the retinal basal lamina by intraocular injection of collagenase. The basal lamina regenerated after chasing the collagenase with Matrigel, which served as a collagenase inhibitor. The basal lamina was reconstituted within 6 h. However, the regenerated basal lamina was located deeper in the retina than normal by reconstituting along the retracted neuroepithelial endfeet demonstrating that these endfeet are the preferred site of basal lamina assembly.