Nuclear lamin isoforms differentially contribute to LINC complex-dependent nucleocytoskeletal coupling and whole-cell mechanics.

The ability of a cell to regulate its mechanical properties is central to its function. Emerging evidence suggests that interactions between the cell nucleus and cytoskeleton influence cell mechanics through poorly understood mechanisms. Here we conduct quantitative confocal imaging to show that the loss of A-type lamins tends to increase nuclear and cellular volume while the loss of B-type lamins behaves in the opposite manner. We use fluorescence recovery after photobleaching, atomic force microscopy, optical tweezer microrheology, and traction force microscopy to demonstrate that A-type lamins engage with both F-actin and vimentin intermediate filaments (VIFs) through the linker of nucleoskeleton and cytoskeleton (LINC) complexes to modulate cortical and cytoplasmic stiffness as well as cellular contractility in mouse embryonic fibroblasts (MEFs). In contrast, we show that B-type lamins predominantly interact with VIFs through LINC complexes to regulate cytoplasmic stiffness and contractility. We then propose a physical model mediated by the lamin­LINC complex that explains these distinct mechanical phenotypes (mechanophenotypes). To verify this model, we use dominant negative constructs and RNA interference to disrupt the LINC complexes that facilitate the interaction of the nucleus with the F-actin and VIF cytoskeletons and show that the loss of these elements results in mechanophenotypes like those observed in MEFs that lack A- or B-type lamin isoforms. Finally, we demonstrate that the loss of each lamin isoform softens the cell nucleus and enhances constricted cell migration but in turn increases migration-induced DNA damage. Together, our findings uncover distinctive roles for each of the four major lamin isoforms in maintaining nucleocytoskeletal interactions and cellular mechanics.

CscoreTool-M infers 3D sub-compartment probabilities within cell population.

MOTIVATION: Computational inference of genome organization based on Hi-C sequencing has greatly aided the understanding of chromatin and nuclear organization in three dimensions (3D). However, existing computational methods fail to address the cell population heterogeneity. Here we describe a probabilistic-modeling-based method called CscoreTool-M that infers multiple 3D genome sub-compartments from Hi-C data. RESULTS: The compartment scores inferred using CscoreTool-M represents the probability of a genomic region locating in a specific sub-compartment. Compared to published methods, CscoreTool-M is more accurate in inferring sub-compartments corresponding to both active and repressed chromatin. The compartment scores calculated by CscoreTool-M also help to quantify the levels of heterogeneity in sub-compartment localization within cell populations. By comparing proliferating cells and terminally differentiated non-proliferating cells, we show that the proliferating cells have higher genome organization heterogeneity, which is likely caused by cells at different cell-cycle stages. By analyzing 10 sub-compartments, we found a sub-compartment containing chromatin potentially related to the early-G1 chromatin regions proximal to the nuclear lamina in HCT116 cells, suggesting the method can deconvolve cell cycle stage-specific genome organization among asynchronously dividing cells. Finally, we show that CscoreTool-M can identify sub-compartments that contain genes enriched in housekeeping or cell-type-specific functions. AVAILABILITY AND IMPLEMENTATION: https://github.com/scoutzxb/CscoreTool-M.

High quality mapping of chromatin at or near the nuclear lamina from small numbers of cells reveals cell cycle and developmental changes of chromatin at the nuclear periphery.

The chromatin associated with the nuclear lamina (NL) is referred to as lamina-associated domains (LADs). Here, we present an adaptation of the tyramide-signal amplification sequencing (TSA-seq) protocol, which we call chromatin pull down-based TSA-seq (cTSA-seq), that can be used to map chromatin regions at or near the NL from as little as 50 000 cells. The cTSA-seq mapped regions are composed of previously defined LADs and smaller chromatin regions that fall within the Hi-C defined B-compartment containing nuclear peripheral heterochromatin. We used cTSA-seq to map chromatin at or near the assembling NL in cultured cells progressing through early G1. cTSA-seq revealed that the distal ends of chromosomes are near or at the reassembling NL during early G1, a feature similar to those found in senescent cells. We expand the use of cTSA-seq to the mapping of chromatin at or near the NL from fixed-frozen mouse cerebellar tissue sections. This mapping reveals a general conservation of NL-associated chromatin and identifies global and local changes during cerebellar development. The cTSA-seq method reported here is useful for analyzing chromatin at or near the NL from small numbers of cells derived from both in vitro and in vivo sources.

The Cdc48-Vms1 complex maintains 26S proteasome architecture.

The 26S proteasome is responsible for most regulated protein turnover and for the degradation of aberrant proteins in eukaryotes. The assembly of this ~2.5 MDa multicatalytic protease requires several dedicated chaperones and, once assembled, substrate selectivity is mediated by ubiquitin conjugation. After modification with ubiquitin, substrates are escorted to the proteasome by myriad factors, including Cdc48 (cell-division cycle 48). Cdc48 also associates with numerous cofactors, but, to date, it is unclear whether each cofactor facilitates proteasome delivery. We discovered that yeast lacking a conserved Cdc48 cofactor, Vms1 [VCP (valosin-containing protein)/Cdc48-associated mitochondrial stress-responsive], accumulate proteasome-targeted ubiquitinated proteins. Vms1 mutant cells also contain elevated levels of unassembled 20S proteasome core particles and select 19S cap subunits. In addition, we found that the ability of Vms1 to support 26S proteasome assembly requires Cdc48 interaction, and that the loss of Vms1 reduced 26S proteasome levels and cell viability after prolonged culture in the stationary phase. The results of the present study highlight an unexpected link between the Cdc48-Vms1 complex and the preservation of proteasome architecture, and indicate how perturbed proteasome assembly affects the turnover of ubiquitinated proteins and maintains viability in aging cells.

BuGZ exhibits guanine nucleotide exchange factor activity toward tubulin.

α- and ß-tubulin form heterodimers, with GTPase activity, that assemble into microtubules. Like other GTPases, the nucleotide-bound state of tubulin heterodimers controls whether the molecules are in a biologically active or inactive state. While α-tubulin in the heterodimer is constitutively bound to GTP, ß-tubulin can be bound to either GDP (GDP-tubulin) or GTP (GTP-tubulin). GTP-tubulin hydrolyzes its GTP to GDP following assembly into a microtubule and, upon disassembly, must exchange its bound GDP for GTP to participate in subsequent microtubule polymerization. Tubulin dimers have been shown to exhibit rapid intrinsic nucleotide exchange in vitro, leading to a commonly accepted belief that a tubulin guanine nucleotide exchange factor (GEF) may be unnecessary in cells. Here, we use quantitative binding assays to show that BuGZ, a spindle assembly factor, binds tightly to GDP-tubulin, less tightly to GTP-tubulin, and weakly to microtubules. We further show that BuGZ promotes the incorporation of GTP into tubulin using a nucleotide exchange assay. The discovery of a tubulin GEF suggests a mechanism that may aid rapid microtubule assembly dynamics in cells.

A Cdc48p-associated factor modulates endoplasmic reticulum-associated degradation, cell stress, and ubiquitinated protein homeostasis.

The hexameric AAA-ATPase, Cdc48p, catalyzes an array of cellular activities, including endoplasmic reticulum (ER)-associated degradation (ERAD), ER/Golgi membrane dynamics, and DNA replication. Accumulating data suggest that unique Cdc48p partners, such as Npl4p-Ufd1p and Ubx1p/Shp1p (p47 in vertebrates), target Cdc48p for these diverse functions. Other Cdc48p-associated proteins have been identified, but the interplay among these factors and their activities is largely cryptic. We now report on a previously uncharacterized Cdc48p-associated protein, Ydr049p, also known as Vms1p, which binds Cdc48p at both the ER membrane and in the cytosol under non-stressed conditions. Loss of YDR049 modestly slows the degradation of the cystic fibrosis transmembrane conductance regulator but does not impede substrate ubiquitination, suggesting that Ydr049p acts at a postubiquitination step in the ERAD pathway. Consistent with Ydr049p playing a role in Cdc48p substrate release, ydr049 mutant cells accumulate Cdc48p-bound ubiquitinated proteins at the ER membrane. Moreover, YDR049 interacts with genes encoding select UBX (ubiquitin regulatory X) and UFD (ubiquitin fusion degradation) proteins, which are Cdc48p partners. Exacerbated growth defects are apparent in some of the mutant combinations, and synergistic effects on the degradation of cystic fibrosis transmembrane conductance regulator and CPY*, which is a soluble ERAD substrate, are evident in specific ydr049-ufd and -ubx mutants. These data suggest that Ydr049p acts in parallel with Cdc48p partners to modulate ERAD and other cellular activities.

An APEX2 proximity ligation method for mapping interactions with the nuclear lamina.

The nuclear lamina (NL) is a meshwork found beneath the inner nuclear membrane. The study of the NL is hindered by the insolubility of the meshwork and has driven the development of proximity ligation methods to identify the NL-associated/proximal proteins, RNA, and DNA. To simplify and improve temporal labeling, we fused APEX2 to the NL protein lamin-B1 to map proteins, RNA, and DNA. The identified NL-interacting/proximal RNAs show a long 3' UTR bias, a finding consistent with an observed bias toward longer 3' UTRs in genes deregulated in lamin-null cells. A C-rich motif was identified in these 3' UTR. Our APEX2-based proteomics identifies a C-rich motif binding regulatory protein that exhibits altered localization in lamin-null cells. Finally, we use APEX2 to map lamina-associated domains (LADs) during the cell cycle and uncover short, H3K27me3-rich variable LADs. Thus, the APEX2-based tools presented here permit identification of proteomes, transcriptomes, and genome elements associated with or proximal to the NL.

A circadian clock regulates sensitivity to cadmium in Paramecium tetraurelia.

The heavy metal cadmium is a dangerous environmental toxicant that can be lethal to humans and other organisms. This paper demonstrates that cadmium is lethal to the ciliated protozoan Paramecium tetraurelia and that a circadian clock modulates the sensitivity of the cells to cadmium. Various concentrations of cadmium were shown to increase the number of behavioral responses, decrease the swimming speed of cells, and generate large vacuole formation in cells prior to death. Cells were grown in either 12-h light/12-h dark or constant dark conditions exhibited a toxic response to 500 microM CdCl(2); the sensitivity of the response was found to vary with a 24-h periodicity. Cells were most sensitive to cadmium at circadian time 0 (CT0), while they were least sensitive in the early evening (CT12). This rhythm persisted even when the cells were grown in constant dark. The oscillation in cadmium sensitivity was shown to be temperature-compensated; cells grown at 18 degrees C and 28 degrees C had a similar 24-h oscillation. Finally, phase shifting experiments demonstrated a phase-dependent response to light. These data establish the criteria required for a circadian clock and demonstrate that P. tetraurelia possesses a circadian-influenced regulatory component of the cadmium toxic response. The Paramecium system is shown to be an excellent model system for the study of the effects of biological rhythms on heavy metal toxicity.

Lamin in inflammation and aging.

Aging is characterized by a progressive loss of tissue function and an increased susceptibility to injury and disease. Many age-associated pathologies manifest an inflammatory component, and this has led to the speculation that aging is at least in part caused by some form of inflammation. However, whether or not inflammation is truly a cause of aging, or is a consequence of the aging process is unknown. Recent work using Drosophila has uncovered a mechanism where the progressive loss of lamin-B in the fat body upon aging triggers systemic inflammation. This inflammatory response perturbs the local immune response of the neighboring gut tissue and leads to hyperplasia. Here, we will discuss the literature connecting lamins to aging and inflammation.

Lamin-B1 contributes to the proper timing of epicardial cell migration and function during embryonic heart development.

Lamin proteins form a meshwork beneath the nuclear envelope and contribute to many different cellular processes. Mutations in lamins cause defective organogenesis in mouse models and human diseases that affect adipose tissue, brain, skeletal muscle, and the heart. In vitro cell culture studies have shown that lamins help maintain nuclear shape and facilitate cell migration. However, whether these defects contribute to improper tissue building in vivo requires further clarification. By studying the heart epicardium during embryogenesis, we show that Lb1-null epicardial cells exhibit in vivo and in vitro migratory delay. Transcriptome analyses of these cells suggest that Lb1 influences the expression of cell adhesion genes, which could affect cell migration during epicardium development. These epicardial defects are consistent with incomplete development of both vascular smooth muscle and compact myocardium at later developmental stages in Lb1-null embryos. Further, we found that Lb1-null epicardial cells have a delayed nuclear morphology change in vivo, suggesting that Lb1 facilitates morphological changes associated with migration. These findings suggest that Lb1 contributes to nuclear shape maintenance and migration of epicardial cells and highlights the use of these cells for in vitro and in vivo study of these classic cell biological phenomena.

Assays to measure ER-associated degradation in yeast.

Endoplasmic reticulum-associated degradation (ERAD) is a process that clears the early secretory pathway of misfolded proteins. Though ERAD is of basic biological importance, the clinical importance of this pathway is emphasized by the fact that mutations that render a protein subject to the ERAD quality control pathway underlie the cause of several diseases. The yeast, Saccharomyces cerevisiae, is a valuable and frequently used model system to study biological processes, such as ERAD, as it is a relatively simple model system for which numerous biochemical and genetic tools are available. In addition, the ERAD system is highly conserved between yeast and man. In this chapter, we describe two methods for the analysis of model substrates that undergo catabolism via the ERAD pathway using S. cerevisiae. In particular, we will describe non-radioactive degradation assays and the analysis of substrate ubiquitylation in vivo with or without the use of ubiquitin overexpression systems. We also describe technical hurdles, which we have encountered in our research, and highlight remedies to overcome them.

Cartilage hair hypoplasia mutations that lead to RMRP promoter inefficiency or RNA transcript instability.

Cartilage hair hypoplasia (CHH; MIM 250250) is an autosomal recessive disease with diverse clinical manifestations. It is caused by mutations in RMRP gene, the RNA component of the ribonucleoprotein complex RNase MRP. Mutations in RMRP have been found in patients in the core promoter region or in the transcribed region, but the pathogenetic effect of the mutations is unclear. Real-time PCR assays confirmed that both promoter (c.-16_-1 dup and c.-15_+2 dup) and transcribed mutations (c.168G > A and c.218A > G) lower the expression level of RMRP. Experiments with 5'RACE, showed that the reduced transcription in the promoter mutants was accompanied by shifting of the transcription initiation sites to nucleotides 5'-upstream of the authentic site. Low levels of RMRP expression levels with transcript mutations were also seen when constructs encoding the wild-type and mutant genes were transfected into cultured cells. The reduced transcription was correlated with greater instability of mutant RMRP transcripts compared to controls. A comparable reduction was seen when a mouse gene containing the c.70A > G mutation (the major mutation in humans with CHH) was introduced into ES cells in place of one of the wild-type alleles. The low expression level of the c.70A > G Rmrp RNA was confirmed by expression assays into cultured cells, and was again correlated with RNA instability. Our results indicate that a loss of mutant RNA transcripts is a critical feature of pathogenesis.