A lamin A/C beta-strand containing the site of lipodystrophy mutations is a major surface epitope for a new panel of monoclonal antibodies

Using a phage-displayed peptide library, we have identified the epitope recognized by a new panel of five monoclonal antibodies (mAbs) raised against full-length recombinant human lamin A. The mAbs were found to recognize both lamin A and C by Western blotting and immunolocalization at the nuclear rim. A nine-amino acid consensus sequence PLLTYRFPP in the common immunoglobulin-like (Ig-like) domain of lamin A/C contains the binding site for all five mAbs. Three-dimensional structure of the Ig-like domain of lamin A/C shows this sequence is a complete beta-strand. This sequence includes arginine-482 (R482) which is mutated in most cases of Dunnigan-type familial partial lipodystrophy (FPLD). R482 may be part of an interaction site on the surface of lamin A/C for lamin-binding proteins associated with lipodystrophy.     

Farnesylated Nuclear Proteins Kugelkern and Lamin Dm0 Affect Nuclear Morphology by Directly Interacting with the Nuclear Membrane

Nuclear shape changes are observed during a variety of developmental processes, pathological conditions, and ageing. The mechanisms underlying nuclear shape changes in the above-mentioned situations have mostly remained unclear. To address the molecular mechanism behind nuclear shape changes, we analyzed how the farnesylated nuclear envelope proteins Kugelkern and lamin Dm0 affect the structure of the nuclear membrane. We found that Kugelkern and lamin Dm0 affect nuclear shape without requiring filament formation or the presence of a classical nuclear lamina. We also could show that the two proteins do not depend on a group of selected inner nuclear membrane proteins for their localization to the nuclear envelope. Surprisingly, we found that farnesylated Kugelkern and lamin Dm0 protein constructs change the morphology of protein-free liposomes. Based on these findings, we propose that farnesylated proteins of the nuclear membrane induce nuclear shape changes by being asymmetrically inserted into the phospholipid bilayer via their farnesylated C-terminal part.     

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.     

Functional domains of the Drosophila bicaudal-D protein

The localization of oocyte-specific determinants in the form of mRNAs to the pro-oocyte is essential for the establishment of oocyte identity. Localization of the Bicaudal-D (Bic-D) protein to the presumptive oocyte is required for the accumulation of Bic-D and other mRNAs to the pro-oocyte. The Bic-D protein contains four well-defined heptad repeat domains characteristic of intermediate filament proteins, and several of the mutations in Bic-D map to these conserved domains. We have undertaken a structure-function analysis of Bic-D by testing the function of mutant Bic-D transgenes (Bic-D(H)) deleted for each of the heptad repeat domains in a Bic-D null background. Our transgenic studies indicate that only the C-terminal heptad repeat deletion results in a protein that has lost zygotic and ovarian functions. The three other deletions result in proteins with full zygotic function, but with affected ovarian function. The functional importance of each domain is well correlated with its conservation in evolution. The analysis of females heterozygous for Bic-D(H) and the existing alleles Bic-D(PA66) or Bic-D(R26) reveals that Bic-D(R26) as well as some of Bic-D(H) transgenes have antimorphic effects. The yeast two-hybrid interaction assay shows that Bic-D forms homodimers. Furthermore, we found that Bic-D exists as a multimeric protein complex consisting of Egl and at least two Bic-D monomers.     

Immunoaffinity purification and functional characterization of interphase and meiotic Drosophila nuclear lamin isoforms

Three isoforms of a single nuclear lamin have been identified in Drosophila. Two, lamins Dm1 and Dm2, are present during interphase and are apparently in equilibrium with each other in vivo. The third, lamin Dmmit, is found in cells that have undergone nuclear envelope breakdown, either during meiosis or mitosis. All three isoforms were purified under nondenaturing conditions using a novel technique of immunoaffinity chromatography and their in vitro activities were examined. Interphase lamins Dm1 and Dm2 can assemble into filaments at physiologic ionic strength; assembly is reversible upon addition of concentrated NaCl. Negative staining of filaments formed in vitro shows long, unbranched bundles approximately 20 nm in diameter. Addition of specific antilamin antibodies blocks in vitro assembly completely. In contrast with lamins Dm1 and Dm2, lamin Dmmit remains soluble at physiologic ionic strength. These observations are consistent with the notion that lamina disassembly in vivo is due, at least in part, to changes in properties of the lamins themselves.     

The different function of single phosphorylation sites of Drosophila melanogaster lamin Dm and lamin C

Lamins' functions are regulated by phosphorylation at specific sites but our understanding of the role of such modifications is practically limited to the function of cdc 2 (cdk1) kinase sites in depolymerization of the nuclear lamina during mitosis. In our study we used Drosophila lamin Dm (B-type) to examine the function of particular phosphorylation sites using pseudophosphorylated mutants mimicking single phosphorylation at experimentally confirmed in vivo phosphosites (S(25)E, S(45)E, T(435)E, S(595)E). We also analyzed lamin C (A-type) and its mutant S(37)E representing the N-terminal cdc2 (mitotic) site as well as lamin Dm R(64)H mutant as a control, non-polymerizing lamin. In the polymerization assay we could observe different effects of N-terminal cdc2 site pseudophosphorylation on A- and B-type lamins: lamin Dm S(45)E mutant was insoluble, in contrast to lamin C S(37)E. Lamin Dm T(435)E (C-terminal cdc2 site) and R(64)H were soluble in vitro. We also confirmed that none of the single phosphorylation site modifications affected the chromatin binding of lamin Dm, in contrast to the lamin C N-terminal cdc2 site. In vivo, all lamin Dm mutants were incorporated efficiently into the nuclear lamina in transfected Drosophila S2 and HeLa cells, although significant amounts of S(45)E and T(435)E were also located in cytoplasm. When farnesylation incompetent mutants were expressed in HeLa cells, lamin Dm T(435)E was cytoplasmic and showed higher mobility in FRAP assay.     

Bicaudal-D is essential for egg chamber formation and cytoskeletal organization in drosophila oogenesis

Bicaudal-D (Bic-D) is required for the transport of determinant mRNAs and proteins to the presumptive oocyte, an essential step in the differentiation of the oocyte. Bic-D protein contains four well-defined heptad repeat domains characteristic of intermediate filament proteins. We characterized the ovarian phenotypes of females expressing mutant Bic-D proteins (Bic-D(H)) deleted for each of the heptad repeat domains. The altered migration of follicle cells we observe in mutant ovaries suggests that Bic-D functions in the germline and directs the inward migration of somatic follicle cells. In the germarium Bic-D is required for the organization of the egg chamber and the structural integrity of the oocyte and nurse cells. Examination of the polarized microtubule network in Bic-D(H) ovaries shows that Bic-D function is required for both the establishment of the polarized microtubule network and its maintenance throughout oogenesis. To explain the multiple functions suggested by the pleiotropic Bic-D phenotype, we propose that Bic-D protein could form itself a filamentous structure and represent an integral, essential part of the cytoskeleton.     

In vivo phosphorylation of Drosophila melanogaster nuclear lamins during both interphase and mitosis

To study phosphorylation of D. melanogaster nuclear lamins in vivo, we used Kc tissue culture cells. Kc cells contain products of both lamin genes, the lamin Dm0 gene encoding constitutive polypeptides expressed in almost all cell types and the developmentally regulated lamin C gene. We grew Kc cells in low phosphate medium and labelled them with (32P(H3PO4. To obtain mitotic cells we used vinblastine to arrest cells in metaphase. Cells were collected, washed, lysed and resultant extracts fractionated in the presence of protein phosphatase inhibitors. D. melanogaster proteins were then denatured by boiling in SDS plus DTT, followed by immunoaffinity chromatography and SDS-PAGE purification. As anticipated, we found that a CNBr fragment derived from the N-terminal part of lamin Dm0-derivatives (amino acid residues 2-158; fragment A) was phosphorylated during both interphase and mitosis. Interphase but not mitotic phosphorylation was found on an internal CNBr fragment (derived from the end of the central rod domain and the first part of the C-terminal lamin tail; amino acid residues 385-548; fragment D). Interphase only phosphorylation was also detected on another CNBr fragment derived from the extreme C-terminal portion of lamin Dm0-derivatives (amino acid residues 549-622; fragment E). To supplement these data, we used 2-D tryptic peptide mapping followed by phosphorImager analysis. We routinely detected at least seven 'spots' derived from interphase lamins but only a single mitotic lamin phosphopeptide.     

Lco1 is a novel widely expressed lamin-binding protein in the nuclear interior

A-type lamins are localized at the nuclear envelope and in the nucleoplasm, and are implicated in human diseases called laminopathies. In a yeast two-hybrid screen with lamin C, we identified a novel widely expressed 171-kDa protein that we named Lamin companion 1 (Lco1). Three independent biochemical assays showed direct binding of Lco1 to the C-terminal tail of A-type lamins with an affinity of 700 nM. Lco1 also bound the lamin B1 tail with lower affinity (2 microM). Ectopic Lco1 was found primarily in the nucleoplasm and colocalized with endogenous intranuclear A-type lamins in HeLa cells. Overexpression of prelamin A caused redistribution of ectopic Lco1 to the nuclear rim together with ectopic lamin A, confirming association of Lco1 with lamin A in vivo. Whereas the major C-terminal lamin-binding fragment of Lco1 was cytoplasmic, the N-terminal Lco1 fragment localized in the nucleoplasm upon expression in cells. Furthermore, full-length Lco1 was nuclear in cells lacking A-type lamins, showing that A-type lamins are not required for nuclear targeting of Lco1. We conclude that Lco1 is a novel intranuclear lamin-binding protein. We hypothesize that Lco1 is involved in organizing the internal lamin network and potentially relevant as a laminopathy disease gene or modifier.     

Null mutants of Drosophila B-type lamin Dm(0) show aberrant tissue differentiation rather than obvious nuclear shape distortion or specific defects during cell proliferation

To elucidate the function of metazoan B-type lamins during development, new null mutations of the Drosophila B-type lamin gene, lamDm(0), were analyzed in parallel with the misg(sz18) mutation, a lamDm(0) allele reported previously. Although in all these mutants, lamin Dm(0) protein was undetectable in neuroblasts and imaginal disc cells from the second instar larval stage onward, cells continued to proliferate. In contrast to the embryonic lethality of another Drosophila lamDm(0) allele, lam(PM15), reported previously, lethality did not occur until late pupal stages. Chromosomal structure and the overall nuclear shape remained normal even at these late pupal stages, although obviously abnormal nuclear pore complex distribution was observed concomitant with the loss of lamin Dm(0) protein. Compensating expression of lamin C was not induced in the absence of lamin Dm(0). Thus, no lamin-containing nuclear structures were found in proliferating larval neuroblasts. We did find that developmental abnormalities appeared in specific organs during the late pupal stage, preceding lethality. Surprisingly, coordinated size increase (hypertrophy) of the ventriculus was observed accompanied by cell division and muscle layer formation. Hypertrophy of the ventriculus correlated with a decrease in ecdysteroid hormone receptor B1 (EcRB1) protein, and furthermore could be suppressed by a heat-inducible EcRB1 transgene. In contrast, both gonadal and CNS tissues exhibited underdevelopment.     

Biosynthesis and interconversion of Drosophila nuclear lamin isoforms during normal growth and in response to heat shock

Two major immunocross-reactive polypeptides of the Drosophila nuclear envelope, distinguishable in interphase cells on the basis of one-dimensional SDS-PAGE mobility, have been localized to the nuclear lamina by immunoelectron microscopy. These have been designated lamins Dm1 and Dm2. Both lamins are apparently derived posttranslationally from a single, primary translation product, lamin Dm0. A pathway has been established whereby lamin Dm0 is processed almost immediately upon synthesis in the cytoplasm to lamin Dm1. Processing occurs posttranslationally, is apparently proteolytic, and has been reconstituted from cell-free extracts in vitro. Processing in vitro is ATP dependent. Once assembled into the nuclear envelope, a portion of lamin Dm1 is converted into lamin Dm2 by differential phosphorylation. Throughout most stages of development and in Schneider 2 tissue culture cells, both lamin isoforms are present in approximately equal abundance. However, during heat shock, lamin Dm2 is converted nearly quantitatively into lamin Dm1. Implications for understanding the regulation of nuclear lamina plasticity through normal growth and in response to heat shock are discussed.     

The functions of Klarsicht and nuclear lamin in developmentally regulated nuclear migrations of photoreceptor cells in the Drosophila eye

Photoreceptor nuclei in the Drosophila eye undergo developmentally regulated migrations. Nuclear migration is known to require the perinuclear protein Klarsicht, but the function of Klarsicht has been obscure. Here, we show that Klarsicht is required for connecting the microtubule organizing center (MTOC) to the nucleus. In addition, in a genetic screen for klarsicht-interacting genes, we identified Lam Dm(0), which encodes nuclear lamin. We find that, like Klarsicht, lamin is required for photoreceptor nuclear migration and for nuclear attachment to the MTOC. Moreover, perinuclear localization of Klarsicht requires lamin. We propose that nuclear migration requires linkage of the MTOC to the nucleus through an interaction between microtubules, Klarsicht, and lamin. The Klarsicht/lamin interaction provides a framework for understanding the mechanistic basis of human laminopathies.     

GSK-3beta-regulated interaction of BICD with dynein is involved in microtubule anchorage at centrosome

Microtubule arrays direct intracellular organization and define cellular polarity. Here, we show a novel function of glycogen synthase kinase-3beta (GSK-3beta) in the organization of microtubule arrays through the interaction with Bicaudal-D (BICD). BICD is known to form a complex with dynein-dynactin and to function in the intracellular vesicle trafficking. Our data revealed that GSK-3beta is required for the binding of BICD to dynein but not to dynactin. Knockdown of GSK-3beta or BICD reduced centrosomally focused microtubules and induced the mislocalization of centrosomal proteins. The unfocused microtubules in GSK-3beta knockdown cells were rescued by the expression of the dynein intermediate chain-BICD fusion protein. Microtubule regrowth assays showed that GSK-3beta and BICD are required for the anchoring of microtubules to the centrosome. These results imply that GSK-3beta may function in transporting centrosomal proteins to the centrosome by stabilizing the BICD1 and dynein complex, resulting in the regulation of a focused microtubule organization.     

The Drosophila melanogaster LEM-domain protein MAN1

Here we describe the Drosophila melanogaster LEM-domain protein encoded by the annotated gene CG3167 which is the putative ortholog to vertebrate MAN1. MAN1 of Drosophila (dMAN1) and vertebrates have the following properties in common. Firstly, both molecules are integral membrane proteins of the inner nuclear membrane (INM) and share the same structural organization comprising an N-terminally located LEM motif, two transmembrane domains in the middle of the molecule, and a conserved RNA recognition motif in the C-terminal region. Secondly, dMAN1 has similar targeting domains as it has been reported for the human protein. Thirdly, immunoprecipitations with dMAN1-specific antibodies revealed that this Drosophila LEM-domain protein is contained in protein complexes together with lamins Dm0 and C. It has been previously shown that human MAN1 binds to A- and B-type lamins in vitro. During embryogenesis and early larval development LEM-domain proteins dMAN1 and otefin show the same expression pattern and are much more abundant in eggs and the first larval instar than in later larval stages and young pupae whereas the LEM-domain protein Bocksbeutel is uniformly expressed in all developmental stages. dMAN1 is detectable in the nuclear envelope of embryonic cells including the pole cells. In mitotic cells of embryos at metaphase and anaphase, LEM-domain proteins dMAN1, otefin and Bocksbeutel were predominantly localized in the region of the two spindle poles whereas the lamin B receptor and lamin Dm0 were more homogeneously distributed. Downregulation of dMAN1 by RNA interference (RNAi) in Drosophila cultured Kc167 cells has no obvious effect on nuclear architecture, viability of RNAi-treated cells and the intracellular distribution of the LEM-domain proteins Bocksbeutel and otefin. In contrast, the localization of dMAN1, Bocksbeutel and otefin at the INM is supported by lamin Dm0. We conclude that the dMAN1 protein is not a limiting component of the nuclear architecture in Drosophila cultured cells.     

Mammalian Golgi-associated Bicaudal-D2 functions in the dynein-dynactin pathway by interacting with these complexes

Genetic analysis in Drosophila suggests that Bicaudal-D functions in an essential microtubule-based transport pathway, together with cytoplasmic dynein and dynactin. However, the molecular mechanism underlying interactions of these proteins has remained elusive. We show here that a mammalian homologue of Bicaudal-D, BICD2, binds to the dynamitin subunit of dynactin. This interaction is confirmed by mass spectrometry, immunoprecipitation studies and in vitro binding assays. In interphase cells, BICD2 mainly localizes to the Golgi complex and has properties of a peripheral coat protein, yet it also co-localizes with dynactin at microtubule plus ends. Overexpression studies using green fluorescent protein-tagged forms of BICD2 verify its intracellular distribution and co-localization with dynactin, and indicate that the C-terminus of BICD2 is responsible for Golgi targeting. Overexpression of the N-terminal domain of BICD2 disrupts minus-end-directed organelle distribution and this portion of BICD2 co-precipitates with cytoplasmic dynein. Nocodazole treatment of cells results in an extensive BICD2-dynactin-dynein co-localization. Taken together, these data suggest that mammalian BICD2 plays a role in the dynein- dynactin interaction on the surface of membranous organelles, by associating with these complexes.     

Nuclear membrane vesicle targeting to chromatin in a Drosophila embryo cell-free system.

A Drosophila cell-free system was used to characterize proteins that are required for targeting vesicles to chromatin and for fusion of vesicles to form nuclear envelopes. Treatment of vesicles with 1 M NaCl abolished their ability to bind to chromatin. Binding of salt-treated vesicles to chromatin could be restored by adding the dialyzed salt extract. Lamin Dm is one of the peripheral proteins whose activity was required, since supplying interphase lamin isoforms Dm1, and Dm2 to the assembly extract restored binding. As opposed to the findings in Xenopus, okadaic acid had no effect on vesicle binding. Trypsin digestion of the salt-stripped vesicles eliminated their association with chromatin even in the presence of the dialyzed salt extract. One vesicles attached to chromatin surface, fusion events took place were found to be sensitive to guanosine 5'-[gamma-thio]triphosphate (GTP gamma S). These chromatin-attached vesicles contained lamin Dm and otefin but not gp210. Thus, these results show that in Drosophila there are two populations of nuclear vesicles. The population that interacts first with chromatin contains lamin and otefin and requires both peripheral and integral membrane proteins, whereas fusion of vesicles requires GTPase activity.     

Nuclear lamin proteins: common structures for paracrystalline,filamentous and lattice forms

Nuclear lamins isolated from a variety of sources and classified as Type V intermediate filament (IF) proteins form a variety of closely related filamentous or paracrystalline polymorphs. Furthermore, the nuclear lamina observed in Xenopus oocytes was shown to consist of an approximately orthogonal lattice of filaments. All of the structures observed thus far have axial periodicities of about 25 or 50 nm. In this work it is shown that the packing of the lamin molecules in both the in vivo and the in vitro assemblies can be explained in terms of a single model in which arrays of antiparallel lamin molecules are half-staggered with respect to one another. Such a scheme closely resembles that previously proposed for four-chain complexes in Types I–IV IF proteins. Higher levels of structure, however, differ significantly. A key interaction in the models of the various lamin assemblies is one occurring between similarly directed segments encompassing the highly conserved part of segment IA and that at the C-terminal end of segment 2B. Such an interaction may also prove to be of importance in IF structure.     

High Mobility Group Protein N5 (HMGN5) and Lamina-associated Polypeptide 2α (LAP2α) Interact and Reciprocally Affect Their Genome-wide Chromatin Organization

The interactions of nuclear lamins with the chromatin fiber play an important role in regulating nuclear architecture and chromatin function; however, the full spectrum of these interactions is not known. We report that the N-terminal domain of the nucleosome-binding protein HMGN5 interacts with the C-terminal domain of the lamin-binding protein LAP2α and that these proteins reciprocally alter their interaction with chromatin. Chromatin immunoprecipitation analysis of cells lacking either HMGN5 or LAP2α reveals that loss of either protein affects the genome-wide distribution of the remaining partner. Our study identifies a new functional link between chromatin-binding and lamin-binding proteins.