* Max-Planck-Institut für Hirnforschung, Abteilung Neurochemie, D-60528 Frankfurt am Main, Germany;
and Theodor-Bovery-Institut, Lehrstuhl für Genetik der Universität Würzburg, D-97074 Würzburg, Germany
Nuclear lamins are thought to play an important role in disassembly and reassembly of the nucleus during mitosis. Here, we describe a Drosophila lamin Dm0 mutant resulting from a P element insertion into the first intron of the Dm0 gene. Homozygous mutant animals showed a severe phenotype including retardation in development, reduced viability, sterility, and impaired locomotion. Immunocytochemical and ultrastructural analysis revealed that reduced lamin Dm0 expression caused an enrichment of nuclear pore complexes in cytoplasmic annulate lamellae and in nuclear envelope clusters. In several cells, particularly the densely packed somata of the central nervous system, defective nuclear envelopes were observed in addition. All aspects of the mutant phenotype were rescued upon P element-mediated germline transformation with a lamin Dm0 transgene. These data constitute the first genetic proof that lamins are essential for the structural organization of the cell nucleus.
Lamins are the major structural proteins of the nuclear
lamina, which 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 is thought to provide a skeletal support for
the nuclear envelope and to mediate the attachment of the
nuclear envelope to interphase chromatin (Aebi et al., 1986 Sequence comparison and biochemical data indicate that
lamin proteins belong to the intermediate filament gene
superfamily characterized by a central Vertebrates have two main types of lamins, i.e., A-type
and B-type lamins (Gerace et al., 1978 In the invertebrate Drosophila melanogaster, two lamin
genes are known. A recently described lamin C gene is expressed only late during embryonic development and thus
may constitute an analog of vertebrate A type lamins (Bossie and Sanders, 1993 Because of their central role in nuclear function and cell
division, the genetic analysis of lamins has been proven
difficult. Here we report the serendipitous isolation and
characterization of a Drosophila lamin mutant resulting
from a P element insertion into the first intron of the Dm0
gene. Flies homozygous for this mutation show a severe
lamin deficiency resulting in impaired viability, fertility,
and locomotion. Ultrastructural analysis of the mutant
central nervous system indicates that the lamin Dm0 gene
product is essential for the structural integrity of the nuclear envelope and the proper integration of NPCs into
the nuclear membrane. In addition, annulate lamellae,
membranous cisternae containing pore complexes, are enriched in the cytoplasm of the mutant cells.
Fly Stocks
All genetic markers used for P element mutagenesis are described in
Lindsley and Zimm (1992) P Element Insertion Screen
P element mutagenesis for autosomal insertions was performed according
to standard methods as described (Zinsmaier et al., 1994 Northern Blot Analysis
Total RNA was isolated from homozygous and heterozygous lamP mutant
flies and from w1118 and In(2LR)Gla control flies, as described (Sass et al.,
1990). RNA (20 µg/lane) was separated on a 1% agarose/5% (wt/vol)
formaldehyde gel and transferred onto a Pall A nylon membrane (Biodyne, Santa Monica, CA). The filter was hybridized to an in vitro transcribed antisense RNA probe of the lamin Dm0 gene (see Fig. 1 A, probe 1) at 65°C in the presence of 50% (wt/vol) formamide as described (Wismar et al., 1995
Western Blot Analysis
Individual flies were homogenized in 100 µl of sample buffer and heated
for 10 min at 70°C, and 50 µl of each extract was loaded on a 10% SDS-
polyacrylamide gel (Laemmli, 1970 Southern Blot Analysis
Genomic DNA of a single adult fly was restricted with EcoRI, separated
on 0.8% agarose gels, and blotted onto Hybond-N+ membrane (Amersham Life Science, Pittsburgh, PA) by alkaline capillary transfer after incubation of the gels in 0.25 M HCl for 10 min. The blot was hybridized with a
32P-labeled probe derived from the rescue plasmid fragment (see Fig. 1 A,
probe 2) as described (Ultsch et al., 1992 Phenotypic Analysis
The righting response of individual flies was tested by tapping Drosophila
culture bottles containing a single fly on a desk top, thus forcing the animal onto its dorsal side. The time required for it to return to a standing
posture was then recorded.
Light Microscopy
To examine the anatomy of mutant gonads, dissected abdomina of female
flies were fixed in Carnoy solution (60% [vol/vol] ethanol/30% [vol/vol]
CHCl3/10% [vol/vol] acetic acid) for 4 h, dehydrated, embedded in paraffin, and cut into 15-µm sections. Tissue staining was performed with hematoxilin and eosin for 7 min and 3 s, respectively. The motility of wt and
mutant sperm was monitored by visual inspection of slightly squashed, unfixed testis preparations.
Immunofluorescense Microscopy
Anesthetized flies were placed in a fly collar (Ashburner, 1989 Electron Microscopy
Drosophila heads were fixed in 2% (wt/vol) OsO4 in 0.1 M sodium cacodylate buffer (pH 7.4) for 2.5 h at 4°C, washed with 0.1 M sodium cacodylate buffer, dehydrated by a series of ethanol dilutions, and incubated
subsequently in 1,2-propylene oxide, 1,2-propylene oxide/Epon 812 (1:1;
vol/vol), and Epon 812 for 5 min, 30 min, and 16 h, respectively. Epon polymerization was then performed for 24 h at 60°C. Ultramicrotome sections (80 nm) were postfixed with buffered OsO4 and negatively stained
with uranyl-acetate and lead citrate for 12 and 3 min, respectively, using
the Ultrastainer apparatus (LKB instruments). Electron micrographs were
taken with a Zeiss microscope (EM10C; Zeiss Inc.).
For the statistical analysis of cellular differences in morphology, head
sections of young adult flies (1 d after eclosion from the pupal envelope)
were used. Cells surrounding the medulla, lobula, and lobula plate (including some cells of central brain) were inspected in several sections. Each
cell was examined for NPC clusters in tangential and cross sections. In addition, annulate lamellae and defective nuclear envelopes were counted.
Only one of these structural features was scored as positive per cell, with
defective envelopes and annulate lamellae having higher priorities than
nuclear pore clusters. NPC clusters were operationally defined as areas
containing more than five NPCs in a regular close distance. Circumferences of cross-sectioned nuclei, NPC densities, and interpore distances
were determined from electron micrographs of head sections of two wt
and homozygous lamP animals each.
P Element-mediated Rescue Experiments
A genomic fragment containing the entire lamin Dm0 transcription unit was
constructed from the P-lacW rescue plasmid and an additional genomic
PCR fragment of the 5 Identification of a P Element Insertion into the
Lamin Gene
A P element insertion into the first intron of the gene encoding the nuclear membrane protein lamin Dm0 of Drosophila melanogaster was discovered in a genetic screen
designed to isolate mutants of ionotropic glutamate receptor subunits (Schuster et al., 1991 Expression of Lamin Dm0 Is Reduced in Homozygous
Mutant Flies
Flies carrying the lamin P-lacW insertion (lamP) were
balanced for the second chromosome with the inversion
In(2LR)Gla, which contains the easily detectable genetic
marker for glaced eyes (Gla). Homozygous and heterozygous lamP adult flies were investigated for lamin gene expression by Northern and Western blot analysis in comparison to w1118 and In(2LR)Gla control flies.
The Dm0 gene encodes two transcripts of 2.8 and 3.0 kb,
which are differentially expressed during development and
probably originate from alternative polyadenylation (Gruenbaum et al., 1988
A single primary translation product (Dm0) of apparent
molecular mass of 76 kD is synthesized from both transcripts and constitutes the precursor of multiple posttranslationally modified isoforms (Smith et al., 1987 Lamin Insertion Flies Show Delayed
Development, Reduced Viability, Impairment of
Locomotion, and Sterility
The most obvious phenotypic changes in the homozygous
lamP flies concerned locomotion behavior, development
and survival, and fertility. After a prolonged developmental time course (delayed by up to 3 d at culture temperature of 24°C), the few hatching homozygous adult mutants
were unable to fly; only occasionally, small jumps could be
observed. This may reflect differences in penetrance of the
insertion in individual animals, resulting in small variations in lamin protein expression. Homozygous lamP mutants moved more slowly than wt or control flies and displayed "spastic" behavior after losing motor coordination.
To monitor the locomotion impairment we tested the righting response by measuring the time for returning to a normal upright posture from a dorsal position. Fig. 3 A shows
that while heterozygous lamP and wt animals right themselves instantly, the mean righting time of homozygous
lamP flies lasted up to several minutes.
Only a small number (5-10%) of the homozygous lamP
animals survived until adulthood in the balanced mutant
strain. Survival was inversely correlated to the population
density in the culture chamber. In overcrowded culture bottles, very few homozygous mutant flies survived, whereas
a higher proportion of homozygous individuals was found
in less populated cultures. During development, three major periods of lethality were detected in the lamP strain,
which included the embryonic stages (lethality of 20-30%), the pupal stages (lethality of 50-60%), and the eclosion of
the adult fly (lethality of 5-10%). Mutant adult flies died
within 2 wk after eclosion.
Homozygous lamP flies of both sexes were sterile. Mutant female flies showed abnormal ovaries whose anatomy
varied among individuals. Comparative sections of wt and
homozygous mutant ovaries are presented in Fig. 3, B and
C. The number of ovarioles, each consisting of a germarium (g) and the vitellarium containing different egg chamber (ec) stages, was significantly reduced in the mutant,
as was the number of individual egg chambers. Late stages
of oogenesis were rarely detected in the mutant ovaries,
and the ones present showed an abnormal morphology. In
contrast to the large oocytes (oc) typically seen in late egg
chambers of wt animals, oocytes of comparable stages were
shortened in homozygous lamP ovaries. In contrast to the
mutant ovaries, the gonads of male mutant flies showed no
gross morphological changes but a drastic reduction in or
complete loss of sperm motility (data not shown).
Germline Transformation with an Intact Lamin Gene
Rescues the Mutant Phenotype
To prove that the P element insertion in the first intron of
the lamin Dm0 gene is indeed the cause for the pleiotropic
phenotype of homozygous lamP flies, we performed P element-mediated germline transformation with a wt lamin
transgene construct, P-lam+ (Fig. 4 A). A unique MunI restriction site at the end of the first intron (see Materials
and Methods) was exploited to assemble the entire transcription unit from the plasmid rescue fragment and a genomic PCR fragment of the 5
Southern blot analysis with probe 2 (Fig. 1 A) showed
that the original P-lacW insertion was still present in the
Tw2-lamP flies (Fig. 4 B). Control flies (w1118), in which a
polymorphic EcoRI restriction site (Fig. 1 A, E*) is absent,
showed a single hybridization signal at ~9.8 kb (Fig. 4 B,
lane 6). Heterozygous lamP flies gave the same 9.8-kb signal but, in addition, a band at ~6.7 kb resulting from the
P-lacW insertion (Fig. 4 B, lane 1). The 9.8-kb band was
absent in homozygous lamP animals (Fig. 4 B, lane 2). In
the Tw2-lamP transformant strain, a new signal was seen at
~9.3 kb (Fig. 4 B, lane 3); this indicates the insertion of
one transgene copy in the transformant strain. The intensity of this rescue signal increased in both male and female
animals homozygous for the transgene, indicating a location of the transgene on the third or fourth chromosome
(Fig. 4 B, lane 4, and not shown). The band of 9.8 kb characteristic of control flies was absent in rescue flies homozygous for the lamP mutation (Fig. 4 B, lane 5).
Western blot analysis of homozygous rescue flies (Fig.
2 B) showed that the Dm lamin isoforms of apparent molecular masses of 74 and 76 kD were present in similar
amounts as in wt, control, and heterozygous mutant strains.
Thus, the defect was indeed rescued at the molecular level.
Consistent with this recovery of protein expression, the
homozygous rescue animals developed normally, were
fully viable, fertile in both sexes, and showed wt locomotion behavior (Fig. 3 A). No obvious phenotypic differences could be found between animals heterozygous and
homozygous for the transgenic P-lam+ insertion (not shown).
Lamin Dmo Immunoreactivity Is Reduced in
Perikarya-rich Regions of the Homozygous lamP
Mutant Brain
To examine whether the reduction of lamin immunoreactivity found by Western blot analysis was due to organspecific deficits in Dm0 gene expression, heads and thoraces of adult homozygous lamP mutants were inspected
by immunofluorescence microscopy using the Dm0 specific monoclonal antibody ADL67 (Riemer et al., 1995
Analysis at higher magnification revealed an altered nuclear distribution of lamin Dm0 in neuronal cells of homozygous lamP mutant flies. While the ADL67 immunofluorescence nicely delineated the surface of wt nuclei
(Fig. 5 E), the residual lamin Dm0 staining of homozygous
mutant nuclei was often fuzzy and irregular (Fig. 5 F).
Similar results (not shown) were also obtained with the monoclonal lamin Dm0 antibodies U25 and T50 (Risau et
al., 1981 No differences in lamin Dm0 immunoreactivity were
seen in animals heterozygous for the lamP mutation (data
not shown). Similarly, in Tw2-lamP rescue animals, lamin
expression in the perikaryal region of the central nervous
system seemed to be restored to that of wt flies (not
shown). In addition, the morphology of the nuclei stained by lamin Dm0 antibodies or DAPI was indistinguishable
from that found in wt preparations (data not shown).
Ultrastructural Analysis Reveals Incomplete
Nuclear Envelopes, Clustering of NPCs in the Nuclear
Envelope, and an Increased Number of
Annulate Lamellae
To further characterize any morphological changes that
might underlie the altered lamin immunofluorescence pattern seen in the visual system of homozygous lamP heads
we performed electron microscopy. The high density of
neuronal cell bodies in this region facilitated the simultaneous inspection of many nuclei. This ultrastructural analysis of cross sections through the heads of young adult flies
revealed striking abnormalities in the homozygous mutant. These included (a) the clustering of NPCs in the nuclear membrane, (b) a high incidence of annulate lamellae,
and (c) a partial loss or even total absence of the nuclear
envelope. Fig. 6 shows representative images obtained from
sections of the optic lobe region around the medulla, lobula, and lobula plate of adult wt, homo- and heterozygous
lamP mutants, and homozygous Tw2-lamP rescue flies. Already at low magnification, a high frequency of NPC clusters and annulate lamellae was routinely seen in homozygous lamP flies (Fig. 6 B) but not in wt (Fig. 6 A),
heterozygous mutant (Fig. 6 C), or rescue (Fig. 6 D) animals. In addition, incomplete nuclear envelopes were often found in the homozygous mutant. A quantitative summary of the morphological characteristics observed in the
medulla and lobula/lobula plate regions of different wt, homo- and heterozygous lamP mutants, and homozygous
Tw2-lamP rescue flies is given in Table I. The incidence of
NPC clusters, annulate lamellae, and incomplete nuclear
envelopes was increased in the homozygous mutant flies,
with ~67% of the mutant nuclei displaying one or more of
these abnormal structures. It should be emphasized that
due to the mode of analysis, i.e., inspection of distant cross-
sections, the extent of the morphological changes observed
might even represent an underestimate of the existing alterations. A slightly increased incidence of NPC clusters,
but not of annulate lamellae or incomplete nuclear envelopes, was observed in the optic lobe cells of heterozygous
lamP flies. No obvious differences as compared to wt flies
could be detected in the rescued Tw2-lamP animals. The
changes produced by the mutation in the homozygous lamP
animals were not accompanied by gross alterations in nuclear size; the average nuclear circumference (±SD) in
cross sections of wt nuclei was 7.92 ± 1.80 µm (n = 366),
whereas nuclei of lamP flies had an average circumference
of 8.97 ± 2.22 µm (n = 301).
Table I.
NPC Clusters, Annulate Lamellae, and Incomplete Nuclear Envelopes in Cells Surrounding the Medulla and Lobula/
Lobula Plate*
; Krohne and Benavente, 1986
; Gerace and Burke,
1988
; Paddy et al., 1990
). Additional functions of the nuclear lamina may include the proper organization and anchoring of nuclear pore complexes (NPCs;1 Aaronson and
Blobel, 1975
; Aebi et al., 1986
; Goldberg and Allen, 1992
).
During mitosis the lamins also play a crucial role in the disassembly and reassembly of the nuclear envelope (Gerace et al., 1978
; Krohne et al., 1978
; Gerace and Blobel, 1980
).
-helical rod domain
containing heptad repeats (Fisher et al., 1986
; McKeon et al.,
1986
; Franke, 1987
; for review see Fuchs and Weber,
1994
). A lamin-like protein is thought to constitute the
progenitor of the intermediate filament proteins (Weber
et al., 1989a
; Dodemont et al., 1990
; Döring and Stick, 1990
).
Highly specific features of lamins include a nuclear localization signal, a COOH-terminal CaaX sequence (C, cysteine; a, aliphatic; X, any amino acid) and characteristic phosphorylation sites in the NH2-terminal head and COOHterminal tail domains. The nuclear localization signal is
responsible for rapid transport of lamins into the nucleus,
thus preventing cytoplasmic assembly (Loewinger and
McKeon, 1988
). Modification by isoprenylation and carboxymethylation at the CaaX motif targets lamins to the inner nuclear membrane (Loewinger and McKeon, 1988
;
Holtz et al., 1989
; Krohne et al., 1989
; Kitten and Nigg,
1991
). The interaction of lamins with the inner nuclear
membrane may in addition be supported by integral membrane proteins, e.g., the putative lamin receptor p58 (Senior and Gerace, 1988
; Worman et al., 1988
; Bailer et al., 1991
) or the lamina-associated proteins (LAPs; Foisner
and Gerace, 1993
). Hyper- and dephosphorylation processes at specific sites close to the lamin's central rod domain are important for regulating the nuclear envelope
assembly and disassembly during mitosis (Gerace and Blobel, 1980
; Smith and Fisher, 1989
; Heald and McKeon,
1990
; Peter et al., 1990
; for review see Nigg, 1992
). The binding of soluble- and/or vesicle-associated lamin to chromosomes is thought to play a critical role during reassembly
of the nuclear envelope at the end of mitosis (Gerace and
Blobel, 1980
; Benavente and Krohne, 1986
; Burke, 1990
;
Glass and Gerace, 1990
; Höger et al., 1991
) in which the
LAPs may also be involved (Foisner and Gerace, 1993
).
Additionally, lamins seem to be required for DNA replication (Meier et al., 1991
; Moir et al., 1994
; for review see
Hutchison, 1994). However, the precise roles of lamins in
nuclear envelope assembly, chromosome condensation,
and DNA replication remain to be unraveled.
; Gerace and Blobel,
1980
; Krohne and Benavente, 1986
). The developmentally
regulated A-type lamins include the lamin A precursor
lamin A0 and its alternative splice variant lamin C (Fisher
et al., 1986
; McKeon et al., 1986
; Röber et al., 1989
; Lin
and Worman, 1993
), whereas the constitutively expressed
lamins B1 and B2 are products of two distinct genes (Höger et al., 1988
, 1990; Zewe et al., 1991
; Lin and Worman, 1995
). Lamin C, in contrast to lamins A and B, has
no COOH-terminal CaaX motif. In case of lamin A, this
isoprenylation motif is removed by proteolytic trimming
after association with the nuclear lamina (Weber et al.,
1989b
; Hennekes and Nigg, 1994
). While lamins A and C
are soluble during mitosis, the B-type lamins remain membrane associated throughout the cell cycle (Gerace and
Blobel, 1980
; Burke and Gerace, 1986
).
; Riemer et al., 1995
). Like the vertebrate lamin C, Drosophila lamin C lacks a COOH-terminal
isoprenylation motif. In contrast, the early and ubiquitously expressed Dm0 lamin gene localized at position 25F
on the left arm of chromosome 2 encodes a polypeptide
precursor of 621 amino acids containing a COOH-terminal CaaX sequence (Gruenbaum et al., 1988
; Osman et al.,
1990
). Both its constitutive expression and the presence of
the CaaX motif classify lamin Dm0 as the equivalent of
vertebrate lamins B. Proteolytic processing of the Dm0
precursor in the cytoplasm followed by differential phosphorylation in the nucleus generates different mature isoforms (Dm1 and Dm2) that are specifically found in interphase and mitotic nuclei (Smith et al., 1987
; Smith and
Fisher, 1989
).
Materials and Methods
. The null white allele w1118, the transposaseproviding stock ry506 Sb P(ry+
2-3), the inversion In(2LR)Gla used as
balancer, and the wild-type (wt) strain Oregon R were obtained through
the Bloomington and Umëa stock collections. The mlac strain with four
P-lacW elements on the X chromosome was obtained from M. Brandt
(Baylor College of Medicine, Houston, TX) via E. Hafen (University of
Zürich, Switzerland). The following strains were used throughout this study (synonym in bold letters): w1118: control flies (unbalanced): w1118/
w1118(
); +/+; +/+; In (2LR)Gla: balanced control flies: w1118/w1118 (
);
+/In(2LR)Gla, Gla; +/+; lamP: homozygous mutant flies: w1118/w1118(
);
lamP-lacW/lamP-lacW; +/+ and heterozygous/balanced mutant flies: w1118/
w1118(
); lamP-lacW/In(2LR)Gla, Gla; +/+; Tw2-lamP: homozygous rescue
flies: w1118/w1118(
); lamP-lacW/lamP-lacW; P-lam+/P-lam+, and heterozygous
rescue flies: w1118/w1118(
); lamP-lacW/In(2LR)Gla, Gla; P-lam+/+ (P-
lam+); wt: Oregon R.
). P-lacW transposable elements (Bier et al., 1989
) were mobilized from the X chromosome with the transposase-providing ry506 Sb P(ry+
2-3) element at 99B
(Robertson et al., 1988
). Jump male progeny of w1118 background and autosomal P-lacW insertions identified by pigmented eyes were crossed in
batches of 50 g to 150 w1118 females per batch. For plasmid rescue of genomic fragments flanking the insertion site, genomic DNA prepared from
overnight egg lays of the individual batches was restricted with EcoRI, ligated, and transformed into bacteria by electroporation. Plasmid DNA prepared from pooled colonies obtained with the different fly batches was digested with EcoRI and analyzed on Southern blots. Digoxygenin-labeled
genomic probes of the glutamate receptor subunits DGluR-I (Ultsch et
al., 1992
), DGluR-II (Schuster et al., 1991
), and DNMDAR-I (Ultsch et al.,
1993
) were used for hybridization. With the DGluR-II probe localized at
25F1-2 (Schuster et al., 1991
), one positive pool was obtained; individual
lines were established thereof by single crosses followed by Southern blot
analysis. The resulting mutant line carrying a P-lacW element insertion at
25F was balanced with the inversion In(2LR)Gla. Plasmid DNA derived
by plasmid rescue from the positive single line was sequenced using a
31mer oligonucleotide primer complementary to the P element's inverted
repeat sequence (5
-CGACGGGACCACCTTATGTTATTTCATCATG3
). Sequences were analyzed using the HUSAR program package of the Deutsches Krebsforschungszentrum (Heidelberg, Germany). A European Molecular Biology Laboratory DNA library search using the BLAST program revealed identity to sequences of the lamin Dm0 gene (Osman et al.,
1990
).
). The probe was 32P labeled using the Riboprobe system
(Promega Biotech, Madison, WI) according to the manufacturer's protocol. To normalize for relative amounts of total RNA applied, the blots
were rehybridized with a 32P-labeled
-tubulin antisense RNA probe.
Fig. 1.
P-lacW insertion
into the lamin Dm0 gene. (A)
Schematic diagram of the genomic region at position
25F1-2 of the second chromosome covering the lamin
Dm0 and the DGluR-II loci.
Exonic regions of the lamin
gene are indicated by open
boxes below the restriction map. The restriction sites
given are: E, EcoRI; B,
BamHI; S, SalI. E* denotes a
polymorphic EcoRI restriction site present in wt but not
the w1118 strains. The P-lacW
element is inserted into the
first intron of the lamin gene
(solid arrow). A horizontal
arrow indicates the 5 to 3
orientation of the
-galactosidase (lacZ) and miniwhite (m-w) genes, and small
boxes denote the inverted repeats of the P element. The
phage insert, originally isolated for DGluR-II, extends
into the lamin Dm0 gene region and hybridizes to the rescue plasmid derived from pUC sequences and adjacent genomic regions. Hybridization probes used for
Northern (probe 1) and Southern (probe 2) blot analysis are indicated. (B) Precise location of the P-lacW insertion in the Dm0 genomic
sequence. The P element is inserted into the center of the first intron of the lamin gene. Open boxes and capital letters indicate exonic
regions. The translation start site is found in the second exon.
[View Larger Version of this Image (22K GIF file)]
). After separation, proteins were
transferred electrophoretically onto nitrocellulose. After blocking in 10%
(wt/vol) nonfat dry milk in PBS overnight at 4°C, the blot was incubated
with hybridoma supernatants of the monoclonal Drosophila lamin Dm0
antibodies U25 (undiluted; kindly provided by H. Saumweber, Humboldt
University, Berlin, Germany; Risau et al., 1981
) and ADL67 (dilution 1:20; kindly provided by N. Stuurman, Biozentrum, Basel, Switzerland; Riemer
et al., 1995
) or of the Drosophila lamin C antibody LC28 (dilution 1:20;
also provided by N. Stuurmann; Riemer et al., 1995
) for 2 h at room temperature. Subsequent incubation with alkaline phosphatase-conjugated
anti-mouse antibodies (Promega Biotech) and visualization with NBT/BCIP
were performed as described (Morr et al., 1995
).
).
), covered
with Tissue Tek (Miles Inc., Kankakee, IN) and frozen at
70°C. Cryosections (10 µm) of fly heads collected on Superfrost Plus microscopy slides
(Menzel, Braunschweig, Germany) were fixed in 4% (wt/vol) formaldehyde for 2 min and washed in PBS, 0.5% (wt/vol) NP-40/PBS, and again
in PBS for 5 min each. After blocking with 5% (vol/vol) goat serum in
PBS for 20 min, the head sections were incubated overnight at room temperature with 1:50 dilutions in PBS of the monoclonal Drosophila lamin
Dm0 antibodies ADL67 (Riemer et al., 1995
), U25 or T50 (Risau et al.,
1981
), or the Drosophila lamin C antibody LC28 (Riemer et al., 1995
). After three washes with PBS for 3 min, the slides were incubated with a 1:500
dilution in PBS of secondary mouse antibodies conjugated to the fluorescent dye Cy3 (Dianova GmbH, Hamburg, Germany) for 30 min in the
dark. After washing the slides twice with PBS for 15 min, DNA staining
was performed with DAPI (1 mg/ml) in the dark. The sections were then
rinsed with deionized water and mounted in glycerol/Mowiol (BASF, Ludwigshofen, Germany). Double fluorescence for lamin and DNA was examined in a fluorescence microscope (Axiophot; Zeiss Inc., Oberkochen,
Germany).
gene region absent from the rescue fragment. The
latter was generated on genomic Drosophila DNA using the oligonucleotide primers 5
-AAGGATCCAAAAACAGCGCAGAGCA-3
(sense)
and 5
-CGTGAGATTTTTGTGACTGA-3
(antisense) at an annealing temperature of 55°C as described (Schuster et al., 1991
). Positions 8 of the
sense and 1 of the antisense primers correspond to positions 1 and 1026 of
the published lamin genomic sequence (Osman et al., 1990
; these sequence data are available from GenBank/EMBL/DDBJ under accession
No. X16275), respectively. A BamHI recognition sequence was added to
the 5
end of the sense primer. The rescue plasmid and the 1033-bp PCR
fragment were restricted with BamHI and MunI and ligated at the MunI
recognition site, yielding the intact lamin gene sequence, with additional
5
and 3
sequences of ~0.25 and 0.9 kb, respectively. For P element-mediated germline transformation, the EcoRI site at the 3
end of this genomic
lamin fragment was blunted by fill in reaction with Klenow enzyme, and the fragment cloned into the BamHI and EcoRV sites of the transformation vector pHS85 (Sass, 1990
) containing an Hsp82 promotor and a neomycin resistance gene as selection marker. The transformation construct (P-lam+) was coinjected with the transposase-providing helper plasmid pp25.7wc (Karess and Rubin, 1984
) into heterozygous lamP preblastoderm embryos according to standard procedures (Rubin and Spradling,
1982
). Hatching adults were crossed with heterozygous lamP flies, and the
F2 progeny was selected on G418 (Sigma Chemical Co., St. Louis, MO)
containing food. Two G418-resistant transformants, Tm1-lamP and Tw2lamP, were obtained. The transformant Tw2-lamP with a P-lam+ element
insertion on the third chromosome was used throughout these studies.
Results
; Ultsch et al., 1992
,
1993
). The lamin gene insertion was identified by hybridization of a
phage insert containing DGluR-II genomic sequences (Schuster, C., and B. Schmitt, unpublished results) to plasmid-rescued DNAs isolated from pools of flies that
carried random insertions of P-lacW elements in their genomes. Sequence analysis of the hybridizing plasmid containing a 4.7-kb rescue fragment revealed a P element insertion after position 710 of the Dm0 gene sequence
(Osman et al., 1990
). This position lies within the first of
three introns (Fig. 1 A) and is located 350 bp upstream of
the translation start site (Fig. 1 B). The genes for both
lamin Dm0 (Gruenbaum et al., 1988
) and the muscle
glutamate receptor subunit DGluR-II (Schuster et al.,
1991
) have been previously localized to position 25F1-2 on
the left arm of the second chromosome. Our analysis
showed that the two genes indeed lie closely together, with
an intergene distance of only ~12 kb (Fig. 1 A).
). Northern hybridizations with an in vitro
synthesized RNA probe encompassing most of the last
exon of the lamin gene (Fig. 1 A, probe 1) are shown in
Fig. 2 A. Both lamin transcripts were barely detectable
(<5% of control) in flies homozygous for the P-lacW insertion; only after extensive overexposure of the radioactively labeled blot, faint signals became visible at 2.8 and
3.0 kb (not shown). In lamP flies heterozygous for the insertion, no obvious differences in transcript levels could be
detected as compared to control flies.
Fig. 2.
Expression of
lamin Dm0 transcripts and
proteins. (A) Northern blot
of total RNA isolated from
adult flies. The top panel shows hybridization with a
lamin-specific riboprobe (Fig.
1 A, probe 1) and the lower
with an -tubulin riboprobe
as control. Lane 1 contains
total RNA of homozygous
w1118, lane 2 of In(2LR)Gla,
lane 3 of heterozygous lamP,
and lane 4 of homozygous
lamP flies. Arrows on the left
indicate the sizes of the two
lamin transcripts derived by
alternative polyadenylation.
Signals for both transcripts
were very faint with the
homozygous mutant strain (lane 4), but clearly visible
after overexposure (not shown). (B) Western blot analysis of homogenates from single adult flies. The blot was probed with the monoclonal antibody U25 recognizing all Dm isoforms (Risau et al., 1981
). Lane 1 contains the homogenate of a wt, lane 2 of a heterozygous
lamP, lane 3 of a homozygous lamP, lane 4 of a homozygous Tw2-lamP, and lane 5 of a homozygous w1118 fly. Both lamin protein bands
were reduced in the homozygous mutant (lane 3) and restored in the rescue fly (lane 4). A cross reacting protein band of apparent molecular mass of 105 kD is not related to the Dm0 gene product and served as an internal control for protein load. Positions of molecular
weight marker proteins are indicated on the left; arrows on the right depict the positions of lamin isoforms.
[View Larger Version of this Image (39K GIF file)]
; Smith and
Fisher, 1989
). During early embryogenesis and in mitosis,
a single soluble isoform of 75 kD (Dmmit) is present. In interphase nuclei, however, the two lamin isoforms Dm1 and Dm2 with apparent molecular masses of 74 and 76 kD, respectively, predominate. Western blot analysis (Fig. 2 B)
with the monoclonal antibody U25 recognizing all isoforms
(Risau et al., 1981
; Smith et al., 1987
) showed a severe reduction of the Dm1 and Dm2 variants in homozygous lamP
flies to <20% of the lamin protein in comparison to wt
(Oregon R) or control (w1118) flies. The 75-kD mitotic isoform could not be observed. Notably, the reduction of the
76-kD band seemed to be more pronounced than that of
the 74-kD Dm1 form in the homozygous mutant. To rule
out a possible crossreaction of the U25 antibody with
Drosophila lamin C (Bossie and Sanders, 1993
; Riemer
et al., 1995
), which exhibits 52% amino acid sequence identity to lamin Dm0 and migrates at a similar position as Dm1
in SDS-polyacrylamide gels, we also used the Dm0-specific
monoclonal antibody ADL67 (Riemer et al., 1995
) for Western blot analysis. A pattern similar to that revealed with
the U25 antibody was again obtained with the ADL67 antibody (data not shown). Both antibodies failed to detect a
reduction in the total amount of lamin protein in heterozygous lamP animals (Fig. 2 B and data not shown). Parallel
Western blots with the lamin C-specific antibody LC28
(Riemer et al., 1995
) failed to reveal detectable differences
in lamin C expression between wt and both homo- and
heterozygous lamP flies (data not shown).
Fig. 3.
Analysis of locomotor behavior and sterility. (A) Righting responses of wt, homo- and heterozygous lamP, and homozygous Tw2lamP flies. Six animals of each genotype were analyzed in six independent measurements. Mean values ± SEM of the observed righting
times are given for each individual. (B and C) Histology of ovaries. Hematoxiline/eosin-stained paraffin sections of the ovaries from a wt
(B) and a homozygous lamP (C) female fly are shown. Note shortened oocytes and reduced number of egg chambers in the bottom
panel. ec, egg chamber; fc, follicle cell; g, germarium; gt, gut; nc, nurse cell; oc, oocyte. The scale bar in B represents 50 µm and is also
valid for C.
[View Larger Versions of these Images (182 + 19K GIF file)]
genomic region. The lamin gene construct begins 251 bp upstream of the transcription
start site, includes the putative TATA box located 29 bp
upstream from the transcription start site, and ends about
0.9 kb downstream of the polyadenylation site of the 3.0kb lamin transcript (Osman et al., 1990
). The lamin gene
construct was cloned into the P element transformation
vector pHS85 that has a neomycin resistance as selection
marker and provides the hsp82 promoter, which is constitutively active (Sass, 1990
). After germline transformation, two neomycin-resistant transformant lines, Tm1-lamP and
Tw2-lamP, were obtained, in which the mutant phenotype
of the homozygous lamP fly was rescued. The transformant
line Tw2-lamP was used throughout the experiments described below.
Fig. 4.
Germline transformation with a lamin transgene. (A) Schematic diagram of the P element
construct used for transformation. The Dm0 lamin transgene was inserted into the P
transposable vector pHS85.
Exonic regions of the lamin
Dm0 gene (lam) are shown as black boxes. Restriction sites
used for lamin transgene
construction (see Materials
and Methods) are indicated
below: B, BamHI; M, MunI; EV, EcoRV. The P element transfer
vector pHS85 contains a fusion of hsp82 protein (hatched) and neomycin phosphotransferase (stippled) gene. The hsp82 promoter is
indicated by an arrow. Flanking P element sequences (P) are
shown with their inverted repeats (triangles). (B) Southern blot
analysis of the transformed rescue strain. EcoRI-restricted genomic DNA isolated from single flies was hybridized to a radioactively labeled plasmid rescue fragment (Fig. 1 A, probe 2). DNA
samples were prepared from the following flies: hetero- and (lane
1), homozygous lamP (lane 2), heterozygous Tw2-lamP (one
transgene copy, lane 3; two transgene copies, lane 4), homozygous Tw2-lamP (lane 5), and homozygous w1118 (lane 6). Arrows
on the right indicate the control (9.8 kb), transgene (9.3 kb), and
lamP (6.7 kb) specific hybridization bands.
[View Larger Versions of these Images (81 + 6K GIF file)]
) and compared to tissue from wt flies (Fig. 5). In heads
from the homozygous mutant, lamin Dm0 immunoreactivity
was significantly decreased in nuclei of the densely packed
cell bodies (perikarya-rich region) of the central nervous
system (Fig. 5 B) as compared to heads from wt flies (Fig. 5
A), whereas the intensities of nuclear DNA staining, as revealed by DAPI fluorescence, were comparable. Notably,
lamin C immunoreactivities of the same perikarya-rich regions were not significantly different between wt (Fig. 5 C) and the lamP (Fig. 5 D) flies. Moreover, the majority of the
neuronal cells strongly immunoreactive with ADL67 showed
no detectable lamin C immunofluorescence in either wt or
mutant animals. Similar differences in lamin Dm0 staining
were also seen in muscle cells of the thoracic region which
express, however, high levels of lamin C in both wt and
lamP mutant. The lamin C expression was comparable to
that of Dm0 seen in wt (data not shown).
Fig. 5.
Indirect immunofluorescence staining of head cryosections by lamin Dm0- and lamin C-specific monoclonal antibodies. Simultaneously processed head sections of a wt (left column) and a homozygous lamP (right column) fly are depicted. The left half column of
each depicts the lamin antibody staining detected by indirect immunofluorescense (IF) and the right half the corresponding DNA staining by DAPI of the same section. A and B show lamin Dm0-specific staining by antibody ADL67 and C and D lamin C-specific staining
by antibody LC28, of a total head hemisection each, with A and C as well as B and D, representing consecutive sections. E and F show
magnifications of selected areas stained with antibody ADL67. Note the altered lamin Dm0 nuclear staining in lamP flies (B and F) as
compared to wt (A and E). Arrowheads in A and B indicate the medulla and lobula/lobula plate cell body regions inspected in the electron-microscopic analysis. Arrows in C and D indicate the same areas displaying low lamin C expression. Anatomical structures of the
fly's central nervous system are indicated in A: cb, central brain; me, medulla; la, lamina; re, retina. The retina displays strong autofluorescence, which is more obvious in wt. Bars: (A-D) 100 µm; (E and F) 10 µm.
[View Larger Version of this Image (137K GIF file)]
). High resolution micrographs in addition suggested alterations in the distribution of DNA staining in
lamP flies. Whereas wt nuclei showed rather homogeneous
DAPI fluorescence, the DNA staining of mutant nuclei
sometimes appeared decompacted and irregular (compare
Fig. 5, E and F).
Fig. 6.
Thin section, electron-microscopic analysis of
cell bodies surrounding the
medulla and lobula/lobula
plate in wt, homo- and heterozygous lamP mutant, and
in Tw2-lamP rescue flies.
Electron micrographs of negatively stained sections are
shown at lower magnification.
A section through several optic lobe cell bodies of the homozygous (ho) lamP mutant
(B) reveals striking differences to wt (A). Arrowheads in B point to examples of annulate lamellae (lower left
cell) and NPC clusters in tangential (middle right cell) and
transversal (lower right cell)
nuclear sections. Similar sections from heterozygous (he)
lamP (C) and homozygous
Tw2-lamP (D) flies show no
obvious differences to wt
(A). Bar, 2 µm.
[View Larger Version of this Image (204K GIF file)]
The most obvious consequence of the lamP genotype
was a high incidence of NPC clusters. Cross sections of wt
nuclei displayed few randomly dispersed NPCs detectable
by a narrowing of the intermembrane space of the nuclear
envelope (Fig. 7, A and E). In homozygous mutants, however, such cross sections frequently contained clustered NPCs at distinct regions of the nuclear envelope (Fig. 7, B
and F). This clustering of NPCs in the homozygous mutant
cells was particularly obvious in tangential sections (Fig. 7
D), while similar sections of nuclei from wt (Fig. 7 C), heterozygous mutant, and rescue (data not shown) animals
carried only a few pore complexes. High resolution images
showed that the NPCs in the homozygous mutant nuclei
were often very densely packed and showed tetragonal symmetry (Fig. 7 H), a feature never found in wt nuclei
(Fig. 7 G). This could not be attributed to major changes
in NPC density resulting from the mutation, since the
mean pore complex density (±SD) was only modestly increased to 2.73 ± 0.60 NPCs/µm nuclear envelope for
lamP (nuclei, n = 296) as compared to a wt value of 2.15 ± 0.67 NPCs/µm nuclear envelope (nuclei, n = 195). Frequency histograms of the interpore distance between individual NPCs confirmed that the major fraction (37%) of the
NPCs in the mutant cells was located within about one pore
diameter distance (0.1-0.2 µm from center to center) from
a neighboring NPC (Fig. 8). Consistently, a significant portion of NPCs is separated by distances >1.0 µm. In contrast, interpore distances in wt nuclei showed the highest
incidence between 0.3-0.4 µm, and distances 1.0 µm
were found to a lesser extent than in mutant nuclei.
The second striking feature of cells from homozygous
lamP mutants was an abundance of annulate lamellae. Annulate lamellae are stacked sheets of membranes in the cytoplasm, which contain a high density of pore complexes
and are often continuous with rough endoplasmic reticulum cisternae (for review see Kessel, 1992). The structure
of pore complexes in annulate lamellae is similar, if not
identical, to that of NPCs in the nuclear envelope. In
~13% of the mutant cells in the perikarya region (Table I), annulate lamellae were found as parallel cisternae apposed
to NPCs of the nuclear envelope (Fig. 9 A) but also as independent curvilinear structures in the cytoplasm (Fig.
9 C). The high resolution micrograph shown in Fig. 9 E
demonstrates the high packing density of NPCs in these
cytoplasmic membranes.
While nearly all cell nuclei from wt, heterozygous mutant, and rescue flies had complete nuclear envelopes, those of homozygous mutants appeared incomplete in ~9% of the total cell population (Table I). Frequently, a fragmentation of the nuclear envelope coincided with the presence of annulate lamellae (Fig. 9 B). A higher magnification of the fragmented envelope is shown in Fig. 9 F. (Due to the absence of nuclear pores, the residual membrane compartments could not always be classified with certainty as nuclear; however, in many cases the membraneous structures surrounded electron-dense material, most probably chromatin). In several cells, a nearly complete loss of the nuclear envelope was found, leaving only a few annulate lamellae-like structures (Fig. 9 D). Changes in the homozygous mutant similar to those described above for the cells surrounding the medulla and lobula/lobula plate were also occasionally seen in retina, lamina, central brain, and muscle (not shown).
The identification of a P element insertion into the first intron of the Dm0 lamin gene reported here constitutes the first successful isolation of a mutation in a gene of the nuclear lamin family. Our data show that reduced Dm0 expression results in a severe mutant phenotype that strongly affects viability, fertility, and locomotion. The observed cellular changes, including NPC clusters, cytoplasmic annulate lamellae, and incomplete nuclear envelopes, indicate that lamin Dm0 is required for normal nuclear envelope assembly and nuclear pore distribution. These data provide clear genetic evidence for the important role of this lamin in the structural organization of the nuclear envelope.
Northern and Western blot analysis of homozygous
lamP flies showed that the P element insertion into the first
intron of the lamin Dm0 gene caused a marked reduction
in the respective transcript and protein levels. Random insertions of P elements into the genome often occur within
regulatory regions and are used in enhancer trap screens
to search for cis-acting elements conferring tissue- or stagespecific expression (O'Kane and Gehring, 1987; Cooley et al.,
1988
; Bier et al., 1989
). It is presently unclear whether such
a regulatory function exists in the first intron of the lamin gene. Alternatively, the P element insertion may affect the
efficiency of pre-mRNA splicing, as reported for transposon insertions in mouse (Pattanakitsakul et al., 1992
; Mülhardt et al., 1994
). Only low levels of correctly processed
Dm0 transcripts of 2.8 and 3.0 kb were found here in homozygous lamP animals. We did not observe, however, a
preferential expression of the 3.0-kb transcript as reported
for adult flies by Gruenbaum et al. (1988)
.
In contrast to the developmentally regulated lamin C
(Riemer et al., 1995), the lamin Dm0 gene is constitutively
expressed from early stages of development onwards; some
decrease in transcript levels is seen during the larval stages
(Gruenbaum et al., 1988
; Riemer at al., 1995). This is consistent with marked effects, in particular high lethality and
delayed morphogenetic maturation, of the insertional
mutation at different stages of development. Only a low
percentage of homozygous mutant animals reached the adult stage. This may reflect variations in mutational penetrance, with comparatively low levels of lamin Dm0 expression being sufficient for survival and/or partial compensation by lamin C (see below). The lack of full lethality
at early developmental stages, in which no expression of
lamin C occurs, may be due to maternal transmission from
the heterozygous mothers. Indeed, lamin Dm0 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 (Frasch et al., 1988
).
Deficits in the oocyte's lamin pool may also explain another characteristic of the lamP phenotype, female sterility.
While sufficient lamin may be provided by heterozygous
mutant mothers, the low amount of Dm0 protein produced
in homozygous mutant females most likely cannot support oocyte development. Together with the observed abnormalities in mutant ovary anatomy, this may be a major cause
of female sterility. Interestingly, the Drosophila lamin Dm0
gene seems to be ubiquitously expressed in all cells analyzed so far with one exception: mature Drosophila sperm
lack lamin Dm0 (Riemer et al., 1995). Male infertility in
homozygous lamP flies thus seems to be caused not by low
sperm lamin contents but rather by an effect of the lamin
Dm0 deficiency on spermatogenesis.
The third phenotypic characteristic of the homozygous
lamP mutant, a severe deficiency in locomotion with delayed righting response and complete loss of flying behavior, suggested a critical role of lamin Dm0 in the neuromuscular system. Immunocytochemistry of homozygous
lamP mutants disclosed a significant reduction of lamin
Dm0 immunofluorescence in both the perikarya region of
the central nervous system, which contains many densely
packed neuronal somata, and muscle cells. Importantly, a
lamin C-specific antibody failed to reveal detectable levels
of lamin C in most Dm0-deficient neuronal nuclei, whereas muscle nuclei showed significant lamin C immunoreactivity in both mutant and wt flies. This suggests that lamin C
may be able to compensate for lamin Dm0 function and is
consistent with the data of Riemer et al. (1995), who reported a high accumulation of lamin Dm0, but not C, transcripts and protein in the central nervous system of late
Drosophila embryos. The same authors also showed that
low lamin C expression persists in the larval eye-antennal discs that give rise to several structures of the visual system. We interpret the striking manifestation of the lamP
phenotype in neurons of the visual system (and other regions of the central nervous system) as a consequence of
the combined effects of both a mutation-induced reduction in lamin Dm0 expression and low lamin C gene activity in these cells. Our high resolution analysis of lamin
Dm0 immunofluorescence indicates that under the latter
conditions not only a reduction in lamin Dm0 content but
also a severe distortion of the regular geometry of the nuclear lamina results.
Additional changes in the structural organization of
neuronal nuclei in homozygous lamP animals were disclosed by electron microscopy. First, we frequently observed incomplete or even missing nuclear envelopes. The
nuclear envelope is disassembled at the onset of open mitosis. During prophase, the nuclear membrane is fragmented into vesicles, and the lamina is depolymerized into
soluble and membrane-bound lamin. Nuclear envelope reassembly takes place in late anaphase and telophase, when
membranes, pore complexes, and lamins reassociate with
chromatin. Several studies have suggested a role of lamins
in targeting nuclear envelope precursor vesicles to chromatin (for review see Lourim and Krohne, 1994). For example, both inclusion into nuclear assembly-competent, cell-free extracts (Burke and Gerace, 1986
; Dabauvalle et
al., 1991
; Ulitzur et al., 1992
) and microinjection in cultured
cells during mitosis (Benavente and Krohne, 1986
) of antilamin antibodies prevent assembly of the nuclear envelope. The observations made here are consistent with
these data and extend the evidence for a critical role of
lamins in envelope formation to the intact organism. It
should, however, be emphasized that in homozygous lamP
flies only a fraction of the inspected cells displayed a partial or total absence of the nuclear envelope membrane (Table I); in most cells, a closed nuclear membrane was seen.
Earlier studies with cell-free extracts from Xenopus eggs
and antibodies specific for the Xenopus B-type lamin XB3,
which was initially thought to be the only lamin expressed
during early developmental stages, have reported formation of a complete membrane including pores around the
nucleus without a lamina present (Newport et al., 1990
; Jenkins et al., 1993
). Therefore, a lamin-independent nuclear membrane assembly pathway was proposed from
these experiments. However, minor amounts (~5-10% of
those of lamin XB3) of another lamin of the B2 type were
subsequently found to be present in these Xenopus extracts (Lourim and Krohne, 1993
). This residual lamin
might have been sufficient to promote nuclear membrane
assembly. Similarly, the small amount of lamin Dm0 still
expressed in homozygous lamP flies might suffice for complete nuclear membrane assembly even in cells that do not
(yet) express lamin C.
The most prominent feature revealed upon ultrastructural analysis was a high abundance of NPC clusters in the
nuclear envelope of homozygous lamP neurons. In 67% of
the nuclei in the perikarya-rich region of the mutant visual
system, NPC clusters were detected, and 37% of all NPCs
were located within 0.1 to 0.2 µm distance from the next
pore complex. The nuclear lamina is thought to organize
and anchor NPCs (Aebi et al., 1986; Stewart and Whytock,
1988
; Goldberg and Allen, 1992
), which are located in
"holes" of the lamina's fibrillar meshwork (Belmont et al.,
1993
). Our data constitute a convincing demonstration
that lamins are indeed essential for the proper spatial organization of NPCs in the nuclear membrane. The latter
might involve either interactions of lamin Dm0 with components of the NPC or its binding to specific proteins in
the inner nuclear membrane. A recent study with lamin
XB3-, but not XB2-depleted, cell-free extract from Xenopus eggs reported an increased formation of regions containing a high density of NPCs on the in vitro assembled
nuclei (Goldberg et al., 1995
). Nuclear pore clustering has
also been observed in mutants of yeast nucleoporin genes
including NUP120, NUP133, NUP145, and NUP159
(Doye et al., 1994
; Wente and Blobel, 1994
; Aitchison et
al., 1995
; Gorsch et al., 1995
; Pemberton et al., 1995
). The
nucleoporin proteins are components of the NPC, and
some of them have been implicated in positioning of the
NPCs. The high incidence of NPC clusters in the homozygous lamP mutant is consistent with a direct interaction between nuclear pore proteins and the lamins. It should be
noted that an uneven distribution of NPCs is often observed in nuclei at early stages of nuclear assembly (Burke
and Gerace, 1986
; Goldberg and Allen, 1992
). Thus, the
NPC clusters found in the lamin-deficient flies may also be
indicative of the disturbed assembly process highlighted by the more severely affected nuclei with incomplete nuclear envelopes.
The third striking feature found in homozygous lamP
mutant cells was an accumulation of annulate lamellae.
These may also be a reflection of early nuclear assembly
stages. Annulate lamellae, membraneous cisternae-containing pore complexes, are frequently found in germ cells
and rapidly dividing somatic cells, like embryonic and tumor cells (for review see Kessel, 1992). They are usually located in the cytoplasm but occasionally also in the nucleoplasm. Viral infection and chemical treatment can enhance
annulate lamellae formation. It has been shown that annulate lamellae do not contain lamins; however, their disassembly and reassembly behavior during mitosis follows
closely that of the nuclear envelope (Cordes et al., 1996
).
Formation of annulate lamellae has been proposed to represent a default pathway, in which pore complexes and
other nuclear membrane components can be stored upon
saturation or absence of chromatin templates (Stafstrom
and Staehelin, 1984
; Meier et al., 1995
). Xenopus cell-free
extracts form annulate lamellae instead of a nuclear envelope when anti-lamin antibodies are added during incubation with external DNA or chromatin (Dabauvalle et al.,
1991
). We interpret, therefore, the high incidence of annulate lamellae in homozygous lamP mutant flies as an accumulation of pore complexes resulting from impaired assembly of the nuclear envelope.
We frequently observed annulate lamellae apposed to
pore clusters in the nuclear envelope; this may be indicative of cytosolic regions specialized for NPC assembly from
soluble components. Another interesting finding of our ultrastructural analysis is that clusters of NPCs at the nuclear surface were often densely packed into crystal-like
structures of tetragonal symmetry, which differs from the
hexagonal symmetry of dense NPC packing most commonly found in annulate lamellae (Scheer and Franke, 1969; Stafstrom and Staehelin, 1984
; Kessel, 1992
). The reason
for this different packing geometry is presently unclear but
may reflect differences in the membrane composition of
annulate lamellae and the nuclear envelope.
The causal relationship between the observed phenotypic and ultrastructural changes and the insertional disruption of the Dm0 gene was demonstrated here by gene rescue. Introduction of a Dm0 transgene harboring the entire transcription unit into lamP flies not only restored all phenotypic features of the wt, but also reversed the ultrastructural changes seen in the mutant. In particular, normalized lamin Dm0 protein levels were paralleled by the reappearance of an intact nuclear membrane, recovery of locomotion and flight behavior, and normal fertility. In addition, whereas eclosion of homozygous lamP pupae was delayed by up to 3 d as compared to control flies, the time course of development was normal in the case of the rescued mutant. Since it appears highly unlikely that the short flanking and intronic sequences contained in the rescue construct in addition to the Dm0 open reading frame correspond to another functional gene, we confidently conclude that the lamP phenotype indeed resulted from reduced lamin Dm0 expression.
The availability of the lamP mutant strain described in this study should foster further genetic and cellular approaches to lamin function. In particular, mobilization of the inserted P element should allow the isolation of novel mutant alleles, including severe deficiencies and null phenotypes. The detailed biochemical and ultrastructural analysis of such mutants may crucially contribute to further deciphering the role(s) of lamin Dm0 proteins in nuclear organization and dynamics. In addition, such mutants may provide a tool to dissect genetically the interactions of lamins with other nuclear envelope and chromatin proteins implicated in the disassembly and reassembly of the nucleus during mitosis.
Received for publication 17 October 1996 and in revised form 18 February 1997.
1. Abbreviations used in this paper: LAP, lamina-associated proteins; NPC, nuclear pore complex; wt, wild type.We thank Dr. H. Saumweber for kindly providing the monoclonal antibodies U25 and T50, Drs. A. Püschel and C. Morgans for critical reading of the manuscript, and Ms. M. Baier, Ms. H. Reitz, and Ms. S. Wartha for secretarial assistance. We also are indebted to Dr. N. Stuurman for kindly providing the monoclonal antibodies ADL67 and LC28 and advice on their use. We are particularly grateful to Dr. G. Krohne (Theodor-BoveriInstitut, University of Würzburg) for help with the interpretation of the ultrastructural data, to Dr. B. Klagges for participating in the mutagenesis screen, to W. Hofer for performing electron microscopy, and to Dr. J.H. Brandstätter for help with ultrastructural analysis and preparation of figures.
This work was supported by Deutsche Forschungsgemeinschaft (Schm 726/4-1) and Fonds der Chemischen Industrie.