To assess the redundancy of lamins B1 and B2, knock-in lines were created that produce lamin B2 from the Lmnb1 locus and lamin B1 from the Lmnb2 locus. Both lines developed severe neurodevelopmental abnormalities, indicating that the abnormalities elicited by the loss of one B-type lamin cannot be prevented by increased synthesis of the other.
Lamins B1 and B2 (B-type lamins) have very similar sequences and are expressed ubiquitously. In addition, both Lmnb1- and Lmnb2-deficient mice die soon after birth with neuronal layering abnormalities in the cerebral cortex, a consequence of defective neuronal migration. The similarities in amino acid sequences, expression patterns, and knockout phenotypes raise the question of whether the two proteins have redundant functions. To investigate this topic, we generated “reciprocal knock-in mice”—mice that make lamin B2 from the Lmnb1 locus (Lmnb1B2/B2) and mice that make lamin B1 from the Lmnb2 locus (Lmnb2B1/B1). Lmnb1B2/B2 mice produced increased amounts of lamin B2 but no lamin B1; they died soon after birth with neuronal layering abnormalities in the cerebral cortex. However, the defects in Lmnb1B2/B2 mice were less severe than those in Lmnb1-knockout mice, indicating that increased amounts of lamin B2 partially ameliorate the abnormalities associated with lamin B1 deficiency. Similarly, increased amounts of lamin B1 in Lmnb2B1/B1 mice did not prevent the neurodevelopmental defects elicited by lamin B2 deficiency. We conclude that lamins B1 and B2 have unique roles in the developing brain and that increased production of one B-type lamin does not fully complement loss of the other.
The B-type lamins are widely assumed to be essential for mammalian cells. In part, this assumption is based on a highly cited study that found that RNAi-mediated knockdown of lamin B1 or lamin B2 in HeLa cells arrested cell growth and led to apoptosis. Studies indicating that B-type lamins play roles in DNA replication, the formation of the mitotic spindle, chromatin organization and regulation of gene expression have fueled the notion that B-type lamins must be essential. But surprisingly, this idea had never been tested with genetic approaches. Earlier this year, a research group from UCLA reported the development of genetically modified mice that lack expression of both Lmnb1 and Lmnb2 in skin keratinocytes (a cell type that proliferates rapidly and participates in complex developmental programs). They reasoned that if lamins B1 and B2 were truly essential, then keratinocyte-specific lamin B1/lamin B2 knockout mice would exhibit severe pathology. Contrary to expectations, the skin and hair of lamin B1/lamin B2-deficient mice were quite normal, indicating that the B-type lamins are dispensable in some cell types. The same UCLA research group has gone on to show that lamin B1 and lamin B2 are critical for neuronal migration in the developing brain and for neuronal survival. The absence of either lamin B1 or lamin B2, or the absence of both B-type lamins, results in severe neurodevelopmental abnormalities.
lamin B1; lamin B2; nuclear envelope; nuclear lamina
Lamin B1 is essential for neuronal migration and progenitor proliferation during the development of the cerebral cortex. The observation of distinct phenotypes of Lmnb1- and Lmnb2-knockout mice and the differences in the nuclear morphology of cortical neurons in vivo suggest that lamin B1 and lamin B2 play distinct functions in the developing brain.
Neuronal migration is essential for the development of the mammalian brain. Here, we document severe defects in neuronal migration and reduced numbers of neurons in lamin B1–deficient mice. Lamin B1 deficiency resulted in striking abnormalities in the nuclear shape of cortical neurons; many neurons contained a solitary nuclear bleb and exhibited an asymmetric distribution of lamin B2. In contrast, lamin B2 deficiency led to increased numbers of neurons with elongated nuclei. We used conditional alleles for Lmnb1 and Lmnb2 to create forebrain-specific knockout mice. The forebrain-specific Lmnb1- and Lmnb2-knockout models had a small forebrain with disorganized layering of neurons and nuclear shape abnormalities, similar to abnormalities identified in the conventional knockout mice. A more severe phenotype, complete atrophy of the cortex, was observed in forebrain-specific Lmnb1/Lmnb2 double-knockout mice. This study demonstrates that both lamin B1 and lamin B2 are essential for brain development, with lamin B1 being required for the integrity of the nuclear lamina, and lamin B2 being important for resistance to nuclear elongation in neurons.
The Dpp/BMP signaling pathway is highly conserved between vertebrates and invertebrates. The recent molecular characterization of the Drosophila crossveinless-2 (cv-2) mutation by Conley and colleagues introduced a novel regulatory step in the Dpp/BMP pathway (Development 127 (2000) 3945). The CV-2 protein is secreted and contains five cysteine-rich (CR) domains similar to those observed in the BMP antagonist Short gastrulation (Sog) of Drosophila and Chordin (Chd) of vertebrates. The mutant phenotype in Drosophila suggests that CV-2 is required for the differentiation of crossvein structures in the wing which require high Dpp levels. Here we present the mouse and human homologs of the Drosophila cv-2 protein. The mouse gene is located on chromosome 9A3 while the human locus maps on chromosome 7p14. CV-2 is expressed dynamically during mouse development, in particular in regions of high BMP signaling such as the posterior primitive streak, ventral tail bud and prevertebral cartilages. We conclude that CV-2 is an evolutionarily conserved extracellular regulator of the Dpp/BMP signaling pathway.
Crossveinless-2; Chordin; Twisted gastrulation; Short gastrulation; BMP; Dpp; Tolloid; CR domain; von Willebrand factor type D domain; TIL domain; Extracellular matrix; Organogenesis; Somite; Sclerotome; Notochord; Neural crest; Dorsal root ganglion; Branchial arch; Cartilage; Vertebra; Intervertebral disc; Lung
Nuclear lamins are major components of the nuclear lamina, and play essential roles in supporting the nucleus and organizing nuclear structures. While a large number of clinically important mutations have been mapped to the LMNA gene in humans, very few mutations have been associated with the B-type lamins. We have shown that lamin B2–deficiency in mice results in severe brain abnormalities. While the early stages of forebrain development in lamin B2–deficient mice appear to be normal, cortical neurons fail to migrate and organize into proper layers within the cerebral cortex. The morphogenesis of the hippocampus and cerebellum is also severely impaired. These phenotypes are reminiscent of lissencephaly, a human brain developmental disorder characterized by an abnormal neuronal migration. Most mutations in lissencephaly patients affect cytoplasmic regulators of nuclear translocation, which is a crucial step in neuronal migration. The phenotypes of lamin B2–deficient mice suggest that lamin B2 may also play a key role in nuclear translocation. Potential mechanisms for lamin B2 involvement, which include mechanical and non-mechanical roles, and participation in LINC complexes in the nuclear envelope, are discussed along with evidence that lamins B1 and B2 play distinct, cell-specific functions.
nuclear envelope; nuclear lamina; lamin B2; LINC complex; nesprin; SUN; lissencephaly; neuronal migration; cortical neurons; nuclear translocation
Lmna yields two major protein products in somatic cells, lamin C and prelamin A. Mature lamin A is produced from prelamin A by four posttranslational processing steps—farnesylation of a carboxyl-terminal cysteine, release of the last three amino acids of the protein, methylation of the farnesylcysteine, and the endoproteolytic release of the carboxyl-terminal 15 amino acids of the protein (including the farnesylcysteine methyl ester). Although the posttranslational processing of prelamin A has been conserved in vertebrate evolution, its physiologic significance remains unclear. Here we review recent studies in which we investigated prelamin A processing with Lmna knock-in mice that produce exclusively prelamin A (LmnaPLAO), mature lamin A (LmnaLAO) or nonfarnesylated prelamin A (LmnanPLAO). We found that the synthesis of lamin C is dispensable in laboratory mice, that the direct production of mature lamin A (completely bypassing all prelamin A processing) causes no discernable pathology in mice, and that exclusive production of nonfarnesylated prelamin A leads to cardiomyopathy.
prelamin A; progeria; restrictive dermopathy; protein farnesylation; cardiomyopathy
Nuclear lamins are major components of the nuclear lamina, and play essential roles in supporting the nucleus and organizing nuclear structures. While a large number of clinically important mutations have been mapped to the LMNA gene in humans, very few mutations have been associated with the B-type lamins. We have shown that lamin B2-deficiency in mice results in severe brain abnormalities. While the early stages of forebrain development in lamin B2-deficient mice appear to be normal, cortical neurons fail to migrate and organize into proper layers within the cerebral cortex. The morphogenesis of the hippocampus and cerebellum is also severely impaired. These phenotypes are reminiscent of lissencephaly, a human brain developmental disorder characterized by an abnormal neuronal migration. Most mutations in lissencephaly patients affect cytoplasmic regulators of nuclear translocation, which is a crucial step in neuronal migration. The phenotypes of lamin B2-deficient mice suggest that lamin B2 may also play a key role in nuclear translocation. Potential mechanisms for lamin B2 involvement, which include mechanical and non-mechanical roles and participation in LINC complexes in the nuclear envelope, are discussed along with evidence that lamins B1 and B2 play distinct, cell-specific functions.
nuclear envelope; nuclear lamina; lamin B2; LINC complex; nesprin; SUN; lissencephaly; neuronal migration; cortical neurons; nuclear translocation
Human geneticists have shown that some progeroid syndromes are caused by mutations that interfere with the conversion of farnesyl-prelamin A to mature lamin A. For example, Hutchinson-Gilford progeria syndrome is caused by LMNA mutations that lead to the accumulation of a farnesylated version of prelamin A. In this review, we discuss the posttranslational modifications of prelamin A and their relevance to the pathogenesis and treatment of progeroid syndromes.
prenylation; ZMPSTE24; restrictive dermopathy; FTI
Crossveinless-2 (Cv2), Twisted Gastrulation (Tsg) and Chordin (Chd) are components of an extracellular biochemical pathway that regulates Bone Morphogenetic Protein (BMP) activity during dorso-ventral patterning of Drosophila and Xenopus embryos, the formation of the fly wing, and mouse skeletogenesis. Because the nature of their genetic interactions remained untested in the mouse, we generated a null allele for Cv2 which was crossed to Tsg and Chd mutants to obtain Cv2;Tsg and Cv2;Chd compound mutants. We found that Cv2 is essential for skeletogenesis as its mutation caused the loss of multiple bone structures and posterior homeotic transformation of the last thoracic vertebra. During early vertebral development, Smad1 phosphorylation in the intervertebral region was decreased in the Cv2 mutant, even though CV2 protein is normally located in the future vertebral bodies. Because Cv2 mutation affects BMP signaling at a distance, this suggested that CV2 is involved in the localization of the BMP morphogenetic signal. Cv2 and Chd mutations did not interact significantly. However, mutation of Tsg was epistatic to all CV2 phenotypes. We propose a model in which CV2 and Tsg participate in the generation of a BMP signaling morphogenetic field during vertebral formation in which CV2 serves to concentrate diffusible Tsg/BMP4 complexes in the vertebral body cartilage.
BMP; Crossveinless-2; Chordin; Twisted Gastrulation; Tolloid; vertebra; morphogenetic field; cartilage; pattern formation
We reported that several HIV protease inhibitors (HIV-PIs) interfere with the endoproteolytic processing of two farnesylated proteins, yeast a-factor and mammalian prelamin A. We proposed that these drugs interfere with prelamin A processing by blocking ZMPSTE24, an integral membrane zinc metalloproteinase known to play a critical role in its processing. However, because all of the drug inhibition studies were performed with cultured fibroblasts or crude membrane fractions rather than on purified enzyme preparations, no definitive conclusions could be drawn. Here, we purified Ste24p, the yeast ortholog of ZMPSTE24, and showed that its enzymatic activity was blocked by three HIV-PIs (lopinavir, ritonavir, and tipranavir). A newer HIV-PI, darunavir, had little effect on Ste24p activity. None of the HIV-PIs had dramatic effects on the enzymatic activity of purified Ste14p, the prenylprotein methyltransferase. These studies strongly support our hypothesis that HIV-PIs block prelamin A processing by directly affecting the enzymatic activity of ZMPSTE24, and in this way they may contribute to lipodystrophy in individuals undergoing HIV-PI treatment.
ZMPSTE24; Ste24p; HIV-PI; lamins; Ste14p; lipodystrophy; protease; methyltransferase
Hutchinson–Gilford progeria syndrome (HGPS) is caused by point mutations that increase utilization of an alternate splice donor site in exon 11 of LMNA (the gene encoding lamin C and prelamin A). The alternate splicing reduces transcripts for wild-type prelamin A and increases transcripts for a truncated prelamin A (progerin). Here, we show that antisense oligonucleotides (ASOs) against exon 11 sequences downstream from the exon 11 splice donor site promote alternate splicing in both wild-type and HGPS fibroblasts, increasing the synthesis of progerin. Indeed, wild-type fibroblasts transfected with these ASOs exhibit progerin levels similar to (or greater than) those in fibroblasts from HGPS patients. This progerin was farnesylated, as judged by metabolic labeling studies. The synthesis of progerin in wild-type fibroblasts was accompanied by the same nuclear shape and gene-expression perturbations observed in HGPS fibroblasts. An ASO corresponding to the 5′ portion of intron 11 also promoted alternate splicing. In contrast, an ASO against exon 11 sequences 5′ to the alternate splice site reduced alternate splicing in HGPS cells and modestly lowered progerin levels. Thus, different ASOs can be used to increase or decrease ‘HGPS splicing’. ASOs represent a new and powerful tool for recreating HGPS pathophysiology in wild-type cells.
Vertebrate Crossveinless-2 (CV2) is a secreted protein that can potentiate or antagonize BMP signaling. Through embryological and biochemical experiments we find that: 1) CV2 functions as a BMP4 feedback inhibitor in ventral regions of the Xenopus embryo; 2) CV2 complexes with Twisted gastrulation and BMP4; 3) CV2 is not a substrate for tolloid proteinases; 4) CV2 binds to purified Chordin protein with high affinity (KD in the 1 nM range); 5) CV2 binds even more strongly to Chordin proteolytic fragments resulting from Tolloid digestion or to full-length Chordin/BMP complexes; 6) CV2 depletion causes the Xenopus embryo to become hypersensitive to the anti-BMP effects of Chordin overexpression or tolloid inhibition. We propose that the CV2/Chordin interaction may help coordinate BMP diffusion to the ventral side of the embryo, ensuring that BMPs liberated from Chordin inhibition by tolloid proteolysis cause peak signaling levels.
Hutchinson-Gilford progeria syndrome (HGPS) is caused by the production of a truncated prelamin A, called progerin, which is farnesylated at its carboxyl terminus. Progerin is targeted to the nuclear envelope and causes misshapen nuclei. Protein farnesyltransferase inhibitors (FTI) mislocalize progerin away from the nuclear envelope and reduce the frequency of misshapen nuclei. To determine whether an FTI would ameliorate disease phenotypes in vivo, we created gene-targeted mice with an HGPS mutation (LmnaHG/+) and then examined the effect of an FTI on disease phenotypes. LmnaHG/+ mice exhibited phenotypes similar to those in human HGPS patients, including retarded growth, reduced amounts of adipose tissue, micrognathia, osteoporosis, and osteolytic lesions in bone. Osteolytic lesions in the ribs led to spontaneous bone fractures. Treatment with an FTI increased adipose tissue mass, improved body weight curves, reduced the number of rib fractures, and improved bone mineralization and bone cortical thickness. These studies suggest that FTIs could be useful for treating humans with HGPS.