scholarly journals Single molecule analysis of lamin dynamics

2018 ◽  
Author(s):  
Leonid A. Serebryannyy ◽  
David A. Ball ◽  
Tatiana S. Karpova ◽  
Tom Misteli
Keyword(s):  
Nuclear Envelope ◽  
Single Molecule ◽  
Dynamic Properties ◽  
Clinical Manifestations ◽  
Lamin A ◽  
Kinetic Properties ◽  
Intermediate Filament Proteins ◽  
Nuclear Lamins ◽  
Cellular Environment ◽  
Associated Proteins

AbstractThe nuclear envelope (NE) is an essential cellular structure that contributes to nuclear stability, organization, and function. Mutations in NE-associated proteins result in a myriad of pathologies with widely diverse clinical manifestations, ages of onsets, and affected tissues. Notably, several hundred disease-causing mutations have been mapped to the LMNA gene, which encodes the intermediate filament proteins lamin A and C, two of the major architectural components of the nuclear envelope. However, how NE dysfunction leads to the highly variable pathologies observed in patient cells and tissues remains poorly understood. One model suggests alterations in the dynamic properties of the nuclear lamina and its associated proteins contribute to disease phenotype. Here, we describe the application of single molecule tracking (SMT) methodology to characterize the behavior of nuclear envelope transmembrane proteins (NETs) and nuclear lamins in their native cellular environment at the single molecule level. As proof-of-concept, we demonstrate by SMT that Halo-tagged lamin B1, Samp1, lamin A, and lamin A Δ50 have distinct binding and kinetic properties, and we identify several disease-relevant mutants which exhibit altered binding dynamics. SMT is also able to separately probe the dynamics of the peripheral and the nucleoplasmic populations of lamin A mutants. We suggest that SMT is a robust and sensitive method to investigate how pathogenic mutations or cellular processes affect protein dynamics at the NE.

scholarly journals Nuclear Lamins a and B1

2000 ◽  
Vol 151 (6) ◽  
pp. 1155-1168 ◽  
Author(s):  
Robert D. Moir ◽  
Miri Yoon ◽  
Satya Khuon ◽  
Robert D. Goldman
Keyword(s):  
Green Fluorescent Protein ◽  
Nuclear Envelope ◽  
Daughter Cell ◽  
Fluorescent Protein ◽  
Lamin A ◽  
Cell Nuclei ◽  
Nuclear Lamins ◽  
Daughter Cells ◽  
Lamin B1 ◽  
Green Fluorescent

At the end of mitosis, the nuclear lamins assemble to form the nuclear lamina during nuclear envelope formation in daughter cells. We have fused A- and B-type nuclear lamins to the green fluorescent protein to study this process in living cells. The results reveal that the A- and B-type lamins exhibit different pathways of assembly. In the early stages of mitosis, both lamins are distributed throughout the cytoplasm in a diffusible (nonpolymerized) state, as demonstrated by fluorescence recovery after photobleaching (FRAP). During the anaphase-telophase transition, lamin B1 begins to become concentrated at the surface of the chromosomes. As the chromosomes reach the spindle poles, virtually all of the detectable lamin B1 has accumulated at their surfaces. Subsequently, this lamin rapidly encloses the entire perimeter of the region containing decondensing chromosomes in each daughter cell. By this time, lamin B1 has assembled into a relatively stable polymer, as indicated by FRAP analyses and insolubility in detergent/high ionic strength solutions. In contrast, the association of lamin A with the nucleus begins only after the major components of the nuclear envelope including pore complexes are assembled in daughter cells. Initially, lamin A is found in an unpolymerized state throughout the nucleoplasm of daughter cell nuclei in early G1 and only gradually becomes incorporated into the peripheral lamina during the first few hours of this stage of the cell cycle. In later stages of G1, FRAP analyses suggest that both green fluorescent protein lamins A and B1 form higher order polymers throughout interphase nuclei.

scholarly journals 112 THE PRESENCE OF LAMIN A/C ANTIGENS IN PORCINE EMBRYOS

2005 ◽  
Vol 17 (2) ◽  
pp. 206
Author(s):  
K. Lee ◽  
W.L. Fodor ◽  
Z. Machaty
Keyword(s):  
Olfactory Bulb ◽  
Nuclear Envelope ◽  
Progenitor Cells ◽  
Nuclear Transfer ◽  
Type A ◽  
Lamin A ◽  
Primary Role ◽  
Nuclear Lamins ◽  
Differentiated Cells

Nuclear lamins are components of the nuclear lamina, and their primary role is to support the nuclear envelope and provide anchorage sites for the chromatin. While type B lamins are expressed in all cells, type A lamins (including lamins A and C) are developmentally regulated and expressed in differentiated cells only. There are conflicting results about the presence of lamin A/C in early mammalian embryos. Lamin A/C was found to localize in the nuclear envelope of bovine, pig, and mouse embryos, while recently it has been reported that early mouse and bovine embryos lacked lamin A/C antigens. It has also been suggested that the existence of lamin A/C in the pronuclei of mouse and bovine nuclear transfer embryos indicated faulty reprogramming. The aim of this study was to investigate the presence of lamin A/C in porcine embryos of different origins (in vivo, parthenogenetic, and nuclear transfer). Embryos of various developmental stages were collected from inseminated gilts. For the production of parthenogenetic embryos, mature oocytes were electroporated and cultured for up to seven days. Fibroblast cells served as differentiated controls; progenitor cells from the olfactory bulb of a porcine fetus were used as undifferentiated controls. Lamin A/C was visualized by immunocytochemistry. Olfactory bulb progenitor cells lacked lamin A/C (0 out of 50 cells showed staining) while all fibroblast nuclei (n = 50) reacted positively with the antibody. GV-stage oocytes, being terminally differentiated cells, also possessed lamin A/C antigens (30/30). Lamin A/C was not detectable in any of the mature oocytes examined (n = 30), but it was found in early cleavage-stage embryos [both in vivo (20/20) and parthenogenetic (30/30)] with the signal becoming weaker in blastocysts (15/15). After nuclear transfer, the lamin A/C signal from fibroblast nuclei disappeared (20/20), consistent with nuclear envelope breakdown. Later it became detectable again; all nuclear transfer embryos reconstructed with either fibroblast or progenitor cells displayed lamin A/C staining in their pronuclei and at all stages examined (n = 65). This suggests that recipient oocytes remodel the donor nuclei and reassemble the nuclear envelopes of both differentiated and undifferentiated cells with type A lamins. Activated oocytes (n = 30) and early embryos (n = 40) were then incubated in the presence of actinomycin D (an inhibitor of RNA polymerase II) or cycloheximide (a protein synthesis inhibitor) for 14 h. Lamin A/C assembly was not perturbed by either treatment, indicating that the assembly did not result from de novo gene transcription but rather from solubilized lamins already in the cytoplasm. The results imply that lamin A/C is present in early pig embryos and that its presence after nuclear transfer is not an indicator of erroneous reprogramming, unlike that reported in cattle and mouse.

The structure and function of the nuclear lamins

1995 ◽  
Vol 53 ◽  
pp. 762-763
Author(s):  
R.D. Goldman ◽  
A. Goldman ◽  
S. Khuon ◽  
M. Montag-Lowy ◽  
R. Moir ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
Nuclear Lamina ◽  
Structure And Function ◽  
Supramolecular Organization ◽  
Intermediate Filament Proteins ◽  
Nuclear Envelope Breakdown ◽  
Nuclear Lamins ◽  
Interphase Cells ◽  
Type V ◽  
And Function

The nuclear lamins are the Type V intermediate filament proteins comprising the nuclear lamina. The lamina is located subjacent to the nucleoplasmic face of the nuclear envelope where it interfaces with chromatin. The lamins are major karyoskeletal proteins which are thought to play important roles in the formation and maintenance of nuclear shape and architecture, as well as in the supramolecular organization of chromatin. The lamins have long been thought to be stable polymeric constituents of the interphase nuclear matrix, due to their insolubility in solutions containing detergents and high salt concentrations. During mitosis, however, the nuclear lamins depolymerize during nuclear envelope breakdown. Subsequently, the lamins repolymerize around the decondensing chromosomes as the nuclear envelope reassembles at the end of mitosis. Although there is a significant amount known about the properties and potential functions of the lamins during mitosis, surprisingly little is known about their properties during interphase. In light of this, we have undertaken experiments which are aimed at determining the properties of the lamins in interphase cells.

The Nuclear Envelope and Human Disease

Physiology ◽  
2004 ◽  
Vol 19 (5) ◽  
pp. 309-314 ◽  
Author(s):  
Antoine Muchir ◽  
Howard J. Worman
Keyword(s):  
Membrane Proteins ◽  
Nuclear Envelope ◽  
Nuclear Membrane ◽  
Intermediate Filament ◽  
Human Disease ◽  
Aging Process ◽  
Intermediate Filament Proteins ◽  
Inner Nuclear Membrane ◽  
Nuclear Lamins ◽  
Nuclear Lamin

Mutations in nuclear lamins A and C, intermediate filament proteins of the nuclear envelope, cause diseases affecting various tissues and the aging process. We review what is known about nuclear lamin function and the different diseases caused by mutations in lamins A and C and associated inner nuclear membrane proteins.

Nuclear envelope disorganization in fibroblasts from lipodystrophic patients with heterozygous R482Q/W mutations in the lamin A/C gene

2001 ◽  
Vol 114 (24) ◽  
pp. 4459-4468 ◽  
Author(s):  
Corinne Vigouroux ◽  
Martine Auclair ◽  
Emmanuelle Dubosclard ◽  
Marcel Pouchelet ◽  
Jacqueline Capeau ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
Primary Cultures ◽  
Ectopic Expression ◽  
Nuclear Pore ◽  
Lamin A ◽  
Nuclear Pore Complexes ◽  
Intermediate Filament Proteins ◽  
Partial Lipodystrophy ◽  
Nuclear Alterations ◽  
Pore Complexes

Dunnigan-type familial partial lipodystrophy (FPLD), characterized by an abnormal body fat redistribution with insulin resistance, is caused by missense heterozygous mutations in A-type lamins (lamins A and C). A- and B-type lamins are ubiquitous intermediate filament proteins that polymerize at the inner face of the nuclear envelope. We have analyzed primary cultures of skin fibroblasts from three patients harboring R482Q or R482W mutations. These cells were euploid and able to cycle and divide. A subpopulation of these cells had abnormal blebbing nuclei with A-type lamins forming a peripheral meshwork, which was frequently disorganized. Inner nuclear membrane protein emerin, an A-type lamin-binding protein, strictly colocalized with this abnormal meshwork. Cells from lipodystrophic patients often had other nuclear envelope defects, mainly consisting of nuclear envelope herniations that were deficient in B-type lamins, nuclear pore complexes, lamina-associated protein 2 beta, and chromatin. The mechanical properties of nuclear envelopes were altered, as judged from the extensive deformations observed in nuclei from heat-shocked cells, and from the low stringency of extraction of their components. These structural nuclear alterations were caused by the lamins A/C mutations, as the same changes were introduced in human control fibroblasts by ectopic expression of R482W mutated lamin A.

scholarly journals The conserved carboxy-terminal cysteine of nuclear lamins is essential for lamin association with the nuclear envelope.

1989 ◽  
Vol 109 (5) ◽  
pp. 2003-2011 ◽  
Author(s):  
G Krohne ◽  
I Waizenegger ◽  
T H Höger
Keyword(s):  
Nuclear Envelope ◽  
Experimental System ◽  
Lamin A ◽  
Xenopus Laevis Oocytes ◽  
Partial Reduction ◽  
Carboxy Terminus ◽  
Nuclear Lamins ◽  
Stable Association ◽  
Carboxy Terminal ◽  
Helical Region

We have analyzed the interaction of soluble nuclear lamins with the nuclear envelope by microinjection of normal and mutated lamins into the cytoplasm of Xenopus laevis oocytes. Our results demonstrate that the conserved cysteine of the carboxy-terminal tetrapeptide Cys Ala/Ser Ile Met of lamins is essential for their association with the nuclear envelope. Removal of this sequence or replacement of the cysteine by serine resulted in Xenopus lamin L1 remaining in a soluble, non-envelope-associated state within the nucleus. Similar mutations of Xenopus lamin A resulted in only partial reduction of nuclear envelope association, indicating that lamin A contains additional signals that can partially compensate for the lack of the cysteine. Mammalian lamin C lacks this tetrapeptide and is not associated with the nuclear envelope in our experimental system. Cloning of the tetrapeptide Cys Ala Ile Met to the carboxy terminus of human lamin C resulted in lamin being found in a nuclear envelope-associated form in oocytes. Mutations at the amino terminus and in the alpha-helical region of lamin L1 revealed that the carboxy terminus mediates the association of lamins with the nuclear envelope; however, this alone is insufficient for maintenance of a stable association with the nuclear envelope.

scholarly journals Membrane-associated phase separation: organization and function emerge from a two-dimensional milieu

2021 ◽  
Author(s):  
Jonathon A Ditlev
Keyword(s):  
Phase Separation ◽  
Cell Biology ◽  
Cellular Environment ◽  
Condensate Formation ◽  
Associated Proteins ◽  
Available Technology ◽  
And Function ◽  
Surrounding Membrane

Abstract Liquid‒liquid phase separation (LLPS) of biomolecules has emerged as an important mechanism that contributes to cellular organization. Phase separated biomolecular condensates, or membrane-less organelles, are compartments composed of specific biomolecules without a surrounding membrane in the nucleus and cytoplasm. LLPS also occurs at membranes, where both lipids and membrane-associated proteins can de-mix to form phase separated compartments. Investigation of these membrane-associated condensates using in vitro biochemical reconstitution and cell biology has provided key insights into the role of phase separation in membrane domain formation and function. However, these studies have generally been limited by available technology to study LLPS on model membranes and the complex cellular environment that regulates condensate formation, composition, and function. Here, I briefly review our current understanding of membrane-associated condensates, establish why LLPS can be advantageous for certain membrane-associated condensates, and offer a perspective for how these condensates may be studied in the future.

scholarly journals The polymerization of actin: II. how nonfilamentous actin becomes nonrandomly distributed in sperm: evidence for the association of this actin with membranes

1976 ◽  
Vol 69 (1) ◽  
pp. 51-72 ◽  
Author(s):  
LG Tilney
Keyword(s):  
Plasma Membrane ◽  
Nuclear Envelope ◽  
Thin Section ◽  
Actin Filaments ◽  
Early Stage ◽  
Mature Sperm ◽  
Vacuole Membrane ◽  
Other Substances ◽  
Associated Proteins ◽  
Basal Half

At an early stage in spermiogenesis the acrosomal vacuole and other organelles including ribosomes are located at the basal end of the cell. From here actin must be transported to its future location at the anterior end of the cell. At no stage in the accumulation of actin in the periacrosomal region is the actin sequestered in a membrane-bounded compartment such as a vacuole or vesicle. Since filaments are not present in the periacrosomal region during the accumulation of the actin even though the fixation of these cells is sufficiently good to distinguish actin filaments in thin section, the actin must accumulate in the nonfilamentous state. The membranes in the periacrosomal region, specifically a portion of the nuclear envelope and the basal half of the acrosomal vacuole membrane, become specialized morphologically in advance of the accumulation of actin in this region. My working hypothesis is that the actin in combination with other substances binds to these specialized membranes and to itself and thus can accumulate in the periacrosmoal region by being trapped on these specialized membranes. Diffusion would then be sufficient to move these substances to this region. In support of this hypothesis are experiments in which I treated mature sperm with detergents, glycols, and hypotonic media, which solubilize or lift away the plasma membrane. The actin and its associated proteins remain attached to these specialized membranes. Thus actin can be nonrandomly distributed in cells in a nonfilamentous state presumably by its association with specialized membranes.

Alterations of nuclear envelope and chromatin organization in mandibuloacral dysplasia, a rare form of laminopathy

2005 ◽  
Vol 23 (2) ◽  
pp. 150-158 ◽  
Author(s):  
Ilaria Filesi ◽  
Francesca Gullotta ◽  
Giovanna Lattanzi ◽  
Maria Rosaria D'Apice ◽  
Cristina Capanni ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
Protein A ◽  
Nuclear Architecture ◽  
Mendelian Inheritance ◽  
Premature Aging ◽  
Lamin A ◽  
Lamin B ◽  
Lamin B Receptor ◽  
Epigenetic Events ◽  
Mandibuloacral Dysplasia

Autosomal recessive mandibuloacral dysplasia [mandibuloacral dysplasia type A (MADA); Online Mendelian Inheritance in Man (OMIM) no. 248370 ] is caused by a mutation in LMNA encoding lamin A/C. Here we show that this mutation causes accumulation of the lamin A precursor protein, a marked alteration of the nuclear architecture and, hence, chromatin disorganization. Heterochromatin domains are altered or completely lost in MADA nuclei, consistent with the finding that heterochromatin-associated protein HP1β and histone H3 methylated at lysine 9 and their nuclear envelope partner protein lamin B receptor (LBR) are delocalized and solubilized. Both accumulation of lamin A precursor and chromatin defects become more severe in older patients. These results strongly suggest that altered chromatin remodeling is a key event in the cascade of epigenetic events causing MADA and could be related to the premature-aging phenotype.

A cDNA from Drosophila melanogaster encodes a lamin C-like intermediate filament protein

1993 ◽  
Vol 104 (4) ◽  
pp. 1263-1272 ◽  
Author(s):  
C.A. Bossie ◽  
M.M. Sanders
Keyword(s):  
Drosophila Melanogaster ◽  
Intermediate Filament ◽  
Blot Analysis ◽  
Polytene Chromosomes ◽  
Intermediate Filament Protein ◽  
Chromosome 2 ◽  
Cross Hybridization ◽  
Intermediate Filament Proteins ◽  
Nuclear Lamins ◽  
Filament Protein

A novel intermediate filament cDNA, pG-IF, has been isolated from a Drosophila melanogaster embryonic expression library screened with a polyclonal antiserum produced against a 46 kDa cytoskeletal protein isolated from Kc cells. This 46 kDa protein is known to be immunologically related to vertebrate intermediate filament proteins. The screen resulted in the isolation of four different cDNA groups. Of these, one has been identified as the previously characterized Drosophila nuclear lamin cDNA, Dm0, and a second, pG-IF, demonstrates homology to Dm0 by cross hybridization on Southern blots. DNA sequence analysis reveals that pG-IF encodes a newly identified intermediate filament protein in Drosophila. Its nucleotide sequence is highly homologous to nuclear lamins with lower homology to cytoplasmic intermediate filament proteins. pG-IF predicts a protein of 621 amino acids with a predicted molecular mass of 69,855 daltons. In vitro transcription and translation of pG-IF yielded a protein with a SDS-PAGE estimated molecular weight of approximately 70 kDa. It contains sequence principles characteristic of class V intermediate filament proteins. Its near neutral pI (6.83) and the lack of a terminal CaaX motif suggests that it may represent a lamin C subtype in Drosophila. In situ hybridization to polytene chromosomes detects one band of hybridization on the right arm of chromosome 2 at or near 51A. This in conjunction with Southern blot analysis of various genomic digests suggests one or more closely placed genes while Northern blot analysis detects two messages in Kc cells.

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