Reimer Stick

Changes in the nuclear lamina composition during early development of Xenopus laevis

Changes in protein composition of the nuclear lamina were monitored during early development in Xenopus. Lamin LIII, the only lamin present in oocyte nuclei, serves as a lamin pool for the formation of pronuclei and early cleavage nuclei. It is present in embryos up to the tail bud stages. Lamins LI and LII, the lamins originally found in adult cell nuclei, appear at characteristic times in development. LI first appears at the midblastula transition (MBT), and LII at the gastrula. Tryptic peptide analysis revealed that all three lamin forms found in the embryo are identical with the adult lamins. De novo synthesis of LIII and LI, observed at MBT, is independent of transcription and must therefore be due to activation of maternal mRNAs. These results are discussed in relation to other nuclear changes occurring during early development.

Filaments made from A- and B-type lamins differ in structure and organization.

Lamins are intermediate filament proteins and the major component of the nuclear lamina. Current views of the lamina are based on the remarkably regular arrangement of lamin LIII in amphibian oocyte nuclei. We have re-examined the LIII lamina and propose a new interpretation of its organization. Rather than consisting of two perpendicular arrays of parallel filaments, we suggest that the oocyte lamina consists of parallel filaments that are interconnected in register to give the impression of a second set of perpendicular filaments. We have also used the oocyte system to investigate the organization of somatic lamins. Currently, it is not feasible to examine the organization of somatic lamins in situ because of their tight association with chromatin. It is also difficult to assemble vertebrate lamin filaments in vitro. Therefore, we have used the oocyte system, where exogenously expressed somatic B-type and A-type lamins assemble into filaments. Expression of B-type lamins induces the formation of intranuclear membranes that are covered by single filament layers. LIII filaments appear identical to the endogenous lamina, whereas lamin B2 assembles into filaments that are organized less precisely. Lamin A induces sheets of thicker filaments on the endogenous lamina and significantly increases the rigidity of the nuclear envelope.

Xenopus Xsal-1, a vertebrate homolog of the region specific homeotic gene spalt of Drosophila.

We have isolated an amphibian homolog of the homeotic gene spalt of Drosophila. Like its Drosophila counterpart the Xenopus Xsal-1 gene encodes a protein that contains three widely separated sets of sequence related double zinc finger motifs of the CC/HH-type as well as a single CC/HH zinc finger. The Xenopus gene encodes a fourth double zinc finger and a single CC/HC zinc finger motif that have no counterpart in the fly protein. Alternative splicing of Xsal-1 transcripts gives rise to RNAs coding for either four, three or two double zinc fingers, respectively. The main expression domains of Xsal-1 in early development are confined to distinct regions along the lateral axon tracts within the midbrain, hindbrain, and spinal cord. Outside the central nervous system Xsal-1 is expressed in the facio-acoustic ganglion and in the developing limb buds. The pattern of expression suggests that Xsal-1 might be under control of signals emanating from the notochord and/or the floor plate and that it might function in neuronal cell specification.

Characterization of the Hydra lamin and its gene; a molecular phylogeny of metazoan lamins.

Abstract. We report sequences for nuclear lamins from the teleost fish Danio and six invertebrates. These include two cnidarians (Hydra and Tealia), one priapulid, two echinoderms, and the cephalochordate Branchiostoma. Combining these results with earlier data on Drosophila, Caenorhabditis elegans, and various vertebrates, the following conclusions on lamin evolution can be drawn. First, all invertebrate lamins resemble in size the vertebrate B-type lamin. Second, all lamins described previously for amphibia, birds and mammals as well as the first lamin of a fish, characterized here, show a cluster of 7 to 12 acidic residues in the tail domain. Since this acidic cluster is absent from all invertebrate lamins including that of the cephalochordate Branchiostoma, it was acquired with the vertebrate lineage. The larger A-type lamin of differentiated cells must have arisen subsequently by gene duplication and insertion of an extra exon. This extra exon of the vertebrate A-lamins is the only major change in domain organization in metazoan lamin evolution. Third, the three introns of the Hydra and Priapulus genes correspond in position to the last three introns of vertebrate B-type lamin genes. Thus the entirely different gene organization of the C. elegans and Drosophila Dmo genes seems to reflect evolutionary drift, which probably also accounts for the fact that C. elegans has the most diverse lamin sequence. Finally we discuss the possibility that two lamin types, a constitutively expressed one and a developmentally regulated one, arose independently on the arthropod and vertebrate lineages.

A role for nuclear lamins in nuclear envelope assembly

The molecular interactions responsible for nuclear envelope assembly after mitosis are not well understood. In this study, we demonstrate that a peptide consisting of the COOH-terminal domain of Xenopus lamin B3 (LB3T) prevents nuclear envelope assembly in Xenopus interphase extracts. Specifically, LB3T inhibits chromatin decondensation and blocks the formation of both the nuclear lamina–pore complex and nuclear membranes. Under these conditions, some vesicles bind to the peripheral regions of the chromatin. These “nonfusogenic” vesicles lack lamin B3 (LB3) and do not bind LB3T; however, “fusogenic” vesicles containing LB3 can bind LB3T, which blocks their association with chromatin and, subsequently, nuclear membrane assembly. LB3T also binds to chromatin in the absence of interphase extract, but only in the presence of purified LB3. Additionally, we show that LB3T inhibits normal lamin polymerization in vitro. These findings suggest that lamin polymerization is required for both chromatin decondensation and the binding of nuclear membrane precursors during the early stages of normal nuclear envelope assembly.

Characterization of NE81, the first lamin-like nucleoskeleton protein in a unicellular organism

Dictyostelium NE81 is the first protein found in a lower eukaryote with properties justifying its denomination as a lamin-like protein. Knockout and overexpression mutants revealed an important role for NE81 in nuclear integrity, chromatin organization, and mechanical stability of cells.

Disappearance and reformation of the nuclear lamina structure during specific stages of meiosis in oocytes

The nuclear lamina is a rigid, proteinaceous layer underlying the inner nuclear membrane of eucaryotic cells. It is present in somatic cell nuclei, disappears during mitosis, and is absent from male meiotic cells. We have investigated the disappearance and reformation of the nuclear lamina during meiosis in oocytes, using immunofluorescence and electron microscopy. We find that the status of the nuclear lamina during meiosis of oocytes differs from the reversible depolymerization seen in mitosis in two respects. First, the lamina disappears during meiotic prophase without affecting the structure of the nuclear membranes or the nuclear pores. Second, the proteins of the dissociated lamina are undetectable by immunological methods in pachytene oocytes, whereas they persist in the cytoplasm during mitosis.

Influence of Lamin A on the Mechanical Properties of Amphibian Oocyte Nuclei Measured by Atomic Force Microscopy

The nuclear lamina is part of the nuclear envelope (NE). Lamin filaments provide the nucleus with mechanical stability and are involved in many nuclear activities. The functional importance of these proteins is highlighted by mutations in lamin genes, which cause a variety of human diseases (laminopathies). Here we describe a method that allows one to quantify the contribution of lamin A protein to the mechanical properties of the NE. Lamin A is ectopically expressed in Xenopus oocytes, where it is incorporated into the NE of the oocyte nucleus, giving rise to a prominent lamina layer at the inner nuclear membrane. Nuclei are then isolated and probed by atomic force microscopy. From the resulting force curves, stiffness values are calculated and compared with those of control nuclei. Expression of lamin A significantly increases the stiffness of oocyte nuclei in a concentration-dependent manner. Since chromatin adds negligibly to nuclear mechanics in these giant nuclei, this method allows one to measure the contribution of individual NE components to nuclear mechanics.

A new model for nuclear lamina organization.

Lamins are intermediate filament proteins that form a network lining the inner nuclear membrane. They provide mechanical strength to the nuclear envelope, but also appear to have many other functions as reflected in the array of diseases caused by lamin mutations. Unlike other intermediate filament proteins, they do not self-assemble into 10 nm filaments in vitro and their in vivo organization is uncertain. We have recently re-examined the organization of a simple B-type lamina in Xenopus oocytes [Goldberg, Huttenlauch, Hutchison and Stick (2008) J. Cell Sci. 121, 215-225] and shown that it consists of tightly packed 8-10 nm filaments with regular cross-connections, tightly opposed to the membrane. When lamin A is expressed in oocytes, it forms organized bundles on top of the B lamina. This has led to a new model for lamina organization which is discussed in the present paper.

The disappearance of the nuclear lamina during spermatogenesis: an electron microscopic and immunofluorescence study.

The nuclear lamina is a proteinaceous layer lying directly beneath the inner nuclear membrane in somatic cells. Here we demonstrate by indirect immunofluorescence and electron microscopy that the lamina is completely absent from the nuclei of spermatocytes and spermatids of the chicken. The absence of a lamina in these cells can also be demonstrated in isolated nuclei lacking the two nuclear membranes. Implications of this finding for possible functions of the nuclear lamina are discussed.