Molecular and Biophysical Characterization of Assembly-Starter Units of Human Vimentin

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Numerous mutations in the humancomprising the major structural components of the nuclear lamina, the fibrous meshwork underlying the inner nuclear membrane (1). They are major determinants of nuclear size and shape and are involved in essential functions such as DNA replication and transcription (1). Lamins A (LA) and C (LC) are alternatively spliced products of the LMNA gene, whereas lamins LB1 and LB2 are encoded by LMNB1 and LMNB2. Structurally, the lamins have a conserved central ␣-helical rod domain flanked by globular head and tail domains. The central rod is responsible for the formation of in-parallel and in-register coiled-coil dimers, the building blocks of lamin polymers. In vitro, lamin dimers form head-to-tail chains, which further interact laterally in an anti-parallel orientation to form highly ordered paracrystals (PCs) (2). However, little is known about the assembly of lamins into higher order structures in vivo.There are Ͼ250 mutations in human LMNA causing a wide range of diseases [for detail, see http//www.umd.be/LMNA/ and (3)]. Among these is the rare premature aging disease, Hutchinson-Gilford progeria syndrome (HGPS). HGPS children typically appear normal at birth, but show growth retardation before the age of 2 years. Further manifestations include loss of hair, lipodystrophy, sclerodermatous skin, osteolysis, and progressive atherosclerosis leading to death at an average age of 13 years due to myocardial infarcts and strokes (4). Most HGPS patients carry the 1824CϾT mutation (G608G), which activates a cryptic splice site resulting in the expression of LA with 50 amino acids deleted near its C terminus (LA⌬50/progerin) (5). As a result, LA⌬50/ progerin remains permanently farnesylated (6, 7), and its accumulation in patients' cells is correlated with the loss of heterochromatin and changes in histone methylation (1).In addition to 1824CϾT, there are 21 other LMNA mutations causing progeria (http://www.umd.be/LMNA/). Some of these are located in the central rod domain, where they could directly impact the assembly of lamins into dimers and higher order structures. Here, we have studied the alterations in nuclear architecture and chromatin organization in one of these mutations, 433GϾA (E145K), located in segment 1B of the central rod domain of LA/C (5). A patient bearing this mutation showed earlier onset cardiovascular defects, only partial loss of hair and ample s.c. fat (5). We show that fibroblasts derived from an E145K patient have severely misshapen nuclei and multiple defects in chromatin organization as reflected by centromere clustering and an abnormal distribution of telomeres throughout the cell cycle. These abnormalities are established during cell division as nuclei assemble in daughter cells. The results demonstrate that the nuclear changes in E145K progeria cells are significantly different from those seen in cells expressing LA⌬50/ progerin, and they also emphasize the essential role of lamins in establishing and maintaining nuclear architecture.

Section: Resultsmentioning

confidence: 99%

A progeria mutation reveals functions for lamin A in nuclear assembly, architecture, and chromosome organization

Taimen

Pfleghaar²,

Shimi

et al. 2009

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215

“…18). To compare mutant vs. WT desmin complex formation, we performed sedimentation velocity ultracentrifugation as described for WT and vimentin fragments (17). Accordingly, WT and 12 of the 14 mutant desmin variants yielded sedimentation curves with sedimentation coefficients, s, ranging between 4.8 S and 5.7 S (mean ϭ 5.2 S, SD Ϯ 0.2), indicating tetramer formation.…”

Section: Resultsmentioning

confidence: 99%

Severe muscle disease-causing desmin mutations interfere with in vitro filament assembly at distinct stages

Bär

Mücke

Kostareva

et al. 2005

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118

Desmin is the major intermediate filament (IF) protein of muscle.Recently, mutations of the desmin gene have been reported to cause familial or sporadic forms of human skeletal, as well as cardiac, myopathy, termed desmin-related myopathy (DRM). The impact of any of these mutations on filament assembly and integration into the cytoskeletal network of myocytes is currently not understood, despite the fact that all cause the same histopathological defect, i.e., desmin aggregation. To gain more insight into the molecular basis of this process, we investigated how mutations within the ␣-helical rod domain of desmin affect both the assembly of the recombinant protein in vitro as well as the filament-forming capacity in cDNA-transfected cells. Whereas 6 of 14 mutants assemble into seemingly normal IFs in the test tube, the other mutants interfere with the assembly process at distinct stages, i.e., tetramer formation, unit-length filament (ULF) formation, filament elongation, and IF maturation. Correspondingly, the mutants with in vitro assembly defects yield dot-like aggregates in transfected cells, whereas the mutants that form IFs constitute a seemingly normal IF cytoskeleton in the cellular context. At present, it is entirely unclear why the latter mutant proteins also lead to aggregate formation in myocytes. Hence, these findings may be a starting point to dissect the contribution of the individual subdomains for desmin pathology and, eventually, the development of therapeutic interventions.desmin-related myopathy ͉ intermediate filaments

“…Initial SAXS measurements were done with solutions of recombinant temperature-sensitive mutant K139C of human vimentin. This mutant variant yields a lower viscosity increase than the WT after assembly initiation at room temperature, because of strongly attenuated longitudinal annealing of its ULFs (16). Therefore we originally used this mutant to avoid possible macroscopic aggregation at relatively high concentrations (up to Ϸ15 mg͞ml) needed for SAXS measurements.…”

Section: Vimentin Assembly In Different Solution Environments As Revementioning

confidence: 99%

“…Recombinant vimentin in 5 mM Tris⅐HCl buffer (pH 8.4) yields predominantly A 11 -type tetramers (16). The filament assembly can be readily initiated by a simultaneous increase of the ionic strength to 50-160 mM and lowering the pH to 7.5 by adding a concentrated buffer͞salt solution (17,18).…”

mentioning

confidence: 99%

“…Vimentin ULFs exhibit some variability of their composition and apparent width but the mean mass value determined by scanning transmission electron microscopy (EM) is close to 1.7 MDa, i.e., 16 dimers or four octamers (19). Cross-linking studies indicated that the dimers within the ULFs are interacting exclusively via the antiparallel, approximately half-staggered A 11 mode (16). Next, the ULFs anneal longitudinally to yield progressively longer filaments.…”

mentioning

confidence: 99%

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Monitoring intermediate filament assembly by small-angle x-ray scattering reveals the molecular architecture of assembly intermediates

Соколова

Kreplak

Wedig

et al. 2006

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103

119

Intermediate filaments (IFs), along with microtubules, microfilaments, and associated cross-bridging proteins, constitute the cytoskeleton of metazoan cells. While crystallographic data on the dimer representing the elementary IF ''building block'' have recently become available, little structural detail is known about both the mature IF architecture and its assembly pathway. Here, we have applied solution small-angle x-ray scattering to investigate the in vitro assembly of a 53-kDa human IF protein vimentin at pH 8.4 by systematically varying the ionic strength conditions, and complemented these experiments by electron microscopy and analytical ultracentrifugation. While a vimentin solution in 5 mM Tris⅐HCl (pH 8.4) contains predominantly tetramers, addition of 20 mM NaCl induces further lateral assembly evidenced by the shift of the sedimentation coeficient and yields a distinct octameric intermediate. Four octamers eventually associate into unit-length filaments (ULFs) that anneal longitudinally. Based on the small-angle x-ray scattering experiments supplemented by crystallographic data and additional structural constraints, 3D molecular models of the vimentin tetramer, octamer, and ULF were constructed. Within each of the three oligomers, the adjacent dimers are aligned exclusively in an approximately half-staggered antiparallel A 11 mode with a distance of 3.2-3.4 nm between their axes. The ULF appears to be a dynamic and a relatively loosely packed structure with a roughly even mass distribution over its cross-section.3D structure ͉ vimentin