Biology subjects

Buchwalter, A.

Publications and source records attributed to Buchwalter, A..

11 recordsLinked to original sources

bioRxiv2026

Cardiomyocyte vulnerability to lamin polymer disruption revealed by saturation mutagenesis

Hundreds of mutations to the broadly expressed LMNA gene cause disease primarily within cardiac, muscular, and adipose tissues (1). Tissue-specific pathogenesis arises when mutant protein dysfunction collides with the unique demands of a specific cell type. Here, we decipher the cell-type-specific consequences of ~15,000 LMNA mutations by completing the first saturation mutagenesis screens in human induced pluripotent cells (hiPSCs) and hiPSC-derived cardiomyocytes using our newly developed single large serine integrase cassette exchange (SLICE) platform. We find that destabilization is a predominant consequence of pathogenic LMNA mutations, is selected against in human populations, and is associated with cardiomyopathy. Mutation sensitivity maps reveal Lamin A quality control at both the subunit and multimer level, resolve lateral and head-to-tail polymerization interfaces, and uncover a convergence between disruption of lamin polymer assembly and pathogenesis. Uniquely in cardiomyocytes, lamin A polymer assembly defects drive profound protein loss, nuclear abnormalities, and cellular toxicity, explaining the origins of cardiac specificity in laminopathy syndromes.

cell biology↗

bioRxiv2026

Emerin loss of function inhibits MyoD-driven differentiation of human iPSCs into skeletal myotubes

Loss of function of the nuclear lamina-associated protein emerin causes Emery-Dreifuss muscular dystrophy (EDMD). Efforts to define emerins essential functions in skeletal muscle have been limited by poor concordance between mouse models and human disease phenotypes. Here, we adapt transgene-driven differentiation of human induced pluripotent stem cells (hiPSCs) into skeletal muscle (iSMs) as a tractable human model for emerin loss of function. We find that EMD knockout (KO) hiPSCs are poorly responsive to combined overexpression of MyoD and Baf60c and produce fewer mature iSMs, indicating that emerin influences muscle differentiation downstream of these differentiation factors. While MyoD acetylation and heterodimerization with E-box proteins are unaffected by emerin loss, MyoD targets including p21 and myogenin are downregulated, and EMD KO cells exhibit impaired cell cycle exit in response to differentiation signals. Transcriptomic analysis of EMD KO iSMs revealed persistent expression of cell cycle genes and decreased expression of terminal muscle differentiation genes. Dysregulated genes do not overlap with lamina-associated domains (LADs) but are instead enriched for targets of polycomb repressive complex 2 (PRC2), which deposits H3K27 trimethylation. Altogether, our data indicate a functional overlap between emerin and PRC2-mediated regulation of terminal muscle differentiation.

cell biology↗

bioRxiv2025

O-GlcNAc modifications regulate lamin A tail processing

Lamin A processing is highly regulated, and necessary for proper assembly of the nuclear lamina facilitating its role in nuclear structure and chromatin organization. Pre-lamin A is first farnesylated, and then a short C-terminal peptide is cleaved to produce mature lamin A. O-GlcNAc Transferase (OGT), a glucose sensitive post-translational modification enzyme, is a potential regulator for lamin A processing. To explore the role of OGT in lamin A biogenesis, we examined the effects of OGT levels and OGT inhibition. Variation in OGT dose or inhibition of its activity did not alter endogenous lamin A abundance or distribution. To more directly test the regulatory effects of O-GlcNAcylation on lamin A, we adapted a tail cleavage assay. Mutation of an OGT binding motif and O-GlcNAc modification sites reduced tail cleavage efficiency, suggesting that O-GlcNAcylation promotes lamin A processing. Our findings add to the understanding of the regulation of lamin A cleavage and identify a potential link between glucose metabolism and lamina biogenesis.

cell biology↗

bioRxiv2025

Lamin B1 and LAP2β resist cytoskeletal force to maintain lamin A/C meshwork organization and preserve nuclear integrity

The nuclear lamins are extremely long-lived proteins in most cell types. As a consequence, lamin function cannot be effectively dissected with temporal precision using standard knock-down approaches. Here, we apply the auxin-inducible degron (AID) system to rapidly deplete each lamin isoform within one cell cycle and reveal the immediate impacts of lamin loss on the nucleus. Surprisingly, neither acute lamin A/C (LA/C), lamin B1 (LB1), nor lamin B2 (LB2) depletion altered nuclear shape or induced nuclear blebbing, indicating that acute lamin loss is not sufficient to alter nuclear morphology. LB1 depletion is immediately followed by LA/C meshwork disorganization due to actin cytoskeletal forces on the lamina, yet neither LA/C nor LB1 depletion induced nuclear rupturing. We found that the abundant inner nuclear membrane protein LAP2{beta} protects nuclear integrity in the absence of LB1, as depletion of both LB1 and LAP2{beta} induced severe LA/C disorganization and frequent nuclear rupturing. Depolymerization of the actin cytoskeleton prevents nuclear rupture in LAP2{beta}- and LB1-depleted nuclei. We conclude that both LB1 and LAP2{beta} resist cytoskeletal force to maintain regular lamin A/C meshwork organization and preserve nuclear integrity.

cell biology↗

bioRxiv2025

C-terminal tagging, transmembrane domain hydrophobicity, and an ER retention motif influence the secretory trafficking of the inner nuclear membrane protein emerin

The inner nuclear membrane (INM), a subdomain of the endoplasmic reticulum (ER), sequesters hundreds of transmembrane proteins within the nucleus. We previously found that one INM protein, emerin, can evade the INM by secretory transport to the lysosome, where it is degraded. In this work, we used targeted mutagenesis to identify intrinsic sequences that promote or inhibit emerins secretory trafficking. By manipulating these sequences across several tag and expression level combinations, we now find that emerins localization is sensitive to C-terminal GFP tagging. While emerins long, hydrophobic C-terminal transmembrane domain facilitates trafficking to the lysosome, extending its lumenal terminus with a GFP tag biases the protein toward this pathway. In contrast, we identify a conserved ER retention sequence that stabilizes N- and C-terminally tagged emerin by limiting its lysosomal flux. These findings underscore long- standing concerns about tagging artifacts and reveal novel determinants of tail-anchored INM protein targeting.

cell biology↗

bioRxiv2024

The nuclear periphery confers repression on H3K9me2-marked genes and transposons to shape cell fate

Heterochromatic loci marked by histone H3 lysine 9 dimethylation (H3K9me2) are enriched at the nuclear periphery in metazoans, but the effect of spatial position on heterochromatin function has not been defined. Here, we remove three nuclear lamins and lamin B receptor (LBR) in mouse embryonic stem cells (mESCs) and show that heterochromatin detaches from the nuclear periphery. Mutant mESCs sustain naive pluripotency and maintain H3K9me2 across the genome but cannot repress H3K9me2-marked genes or transposons. Further, mutant cells fail to differentiate into epiblast-like cells (EpiLCs), a transition that requires the expansion of H3K9me2 across the genome. Mutant EpiLCs can silence naive pluripotency genes and activate epiblast-stage genes. However, H3K9me2 cannot repress markers of alternative fates, including primitive endoderm. We conclude that the nuclear periphery controls the spatial position, dynamic remodeling, and repressive capacity of H3K9me2-marked heterochromatin to shape cell fate decisions.

cell biology↗

bioRxiv2023

Modeling the consequences of age-linked rDNA hypermethylation with dCas9-directed DNA methylation in human cells

Ribosomal DNA (rDNA) genes encode the structural RNAs of the ribosome and are present in hundreds of copies in mammalian genomes. Age-linked DNA hypermethylation throughout the rDNA constitutes a robust "methylation clock" that accurately reports age, yet the consequences of hypermethylation on rDNA function are unknown. We confirmed that pervasive hypermethylation of rDNA occurs during mammalian aging and senescence while rDNA copy number remains stable. We found that DNA methylation is exclusively found on the promoters and gene bodies of inactive rDNA. To model the effects of age-linked methylation on rDNA function, we directed de novo DNA methylation to the rDNA promoter or gene body with a nuclease-dead Cas9 (dCas9) - DNA methyltransferase fusion enzyme in human cells. Hypermethylation at each target site had no detectable effect on rRNA transcription, nucleolar morphology, or cellular growth rate. Instead, human UBF and Pol I remain bound to rDNA promoters in the presence of increased DNA methylation. These data suggest that promoter methylation is not sufficient to impair transcription of the human rDNA and imply that the human rDNA transcription machinery may be resilient to age-linked rDNA hypermethylation.

molecular biology↗

bioRxiv2023

Proteome birthdating reveals age-selectivity of protein ubiquitination

Within a cell, proteins have distinct and highly variable half-lives. As a result, the molecular ages of proteins can range from seconds to years. How the age of a protein influences its environmental interactions is a largely unexplored area of biology. To investigate the age-selectivity of cellular pathways, we developed a methodology termed "proteome birthdating" that barcodes proteins based on their time of synthesis. We demonstrate that this approach provides accurate measurements of protein turnover kinetics without the requirement for multiple kinetic time points. As a first use case of the birthdated proteome, we investigated the age distribution of the human ubiquitinome. Our results indicate that the vast majority of ubiquitinated proteins in a cell consist of newly synthesized proteins and that these young proteins constitute the bulk of the degradative flux through the proteasome. Rapidly ubiquitinated nascent proteins are enriched in cytosolic subunits of large protein complexes. Conversely, proteins destined for the secretory pathway and vesicular transport have older ubiquitinated populations. Our data also identified a smaller subset of very old ubiquitinated cellular proteins that do not appear to be targeted to the proteasome for rapid degradation. Together, our data provide an age census of the human ubiquitinome and establish proteome birthdating as a robust methodology for investigating the protein age-selectivity of diverse cellular pathways. Significance StatementCellular proteins have widely different ages - whereas some have been recently synthesized, others have existed in the cell for days or even years. How a proteins age influences its functions and interactions is largely unknown because it is difficult to globally differentiate proteins based on their time of synthesis. To address this challenge, we developed an analytical method named "proteome birthdating" that can partition cellular proteins into multiple discernible age groups. As an example application, we used proteome birthdating to examine the protein age-selectivity of the ubiquitin proteasome system, a major protein degradation pathway in eukaryotes. Our results show that proteins destined for degradation by this pathway consist of either particularly young or particularly old proteins, with the former being the predominant population. Together, our results establish proteome birthdating as a useful approach for analyzing the turnover of proteins and investigating the functional consequences of their age.

biochemistry↗

bioRxiv2023

Long lifetime and selective accumulation of the A-type lamins accounts for the tissue specificity of Hutchinson-Gilford progeria syndrome

Mutations to the LMNA gene cause laminopathies including Hutchinson-Gilford progeria syndrome (HGPS) that severely affect the cardiovascular system. The origins of tissue specificity in these diseases are unclear, as the A-type Lamins are abundant and broadly expressed proteins. We show that A-type Lamin protein and transcript levels are uncorrelated across tissues. As protein-transcript discordance can be caused by variations in protein lifetime, we applied quantitative proteomics to profile protein turnover rates in healthy and progeroid tissues. We discover that tissue context and disease mutation each influence A-type Lamin protein lifetime. Lamin A/C has a weeks-long lifetime in the aorta, heart, and fat, where progeroid pathology is apparent, but a days-long lifetime in the liver and gastrointestinal tract, which are spared from disease. The A-type Lamins are insoluble and densely bundled in cardiovascular tissues, which may present an energetic barrier to degradation and promote long protein lifetime. Progerin is even more long-lived than Lamin A/C in the cardiovascular system and accumulates there over time. Progerin accumulation interferes broadly with protein homeostasis, as hundreds of abundant proteins turn over more slowly in progeroid tissues. These findings indicate that potential gene therapy interventions for HGPS will have significant latency and limited potency in disrupting the long-lived Progerin protein. Finally, we reveal that human disease alleles are significantly over-represented in the long-lived proteome, indicating that long protein lifetime may influence disease pathology and present a significant barrier to gene therapies for numerous human diseases. Significance statementMany human diseases are caused by mutations to broadly expressed proteins, yet disease mysteriously manifests only in specific tissues. An example of this is Hutchinson-Gilford progeria syndrome (HGPS), which is caused by a mutation to the Lamin A/C protein. We show that this mutation slows the turnover of Lamin A/C proteins in disease-afflicted tissues, causing the mutant "Progerin" protein to accumulate over time and interfere with the normal turnover of hundreds of other proteins. Because Progerin is a long-lived protein, effective therapies for this disease will need to attack the protein and not just the gene that encodes it.

cell biology↗

bioRxiv2022

Combined measurements of proteome and cell turnover reveal the influences of tissue context and pathological aging on protein and organelle lifetimes

The lifespans of proteins can range from minutes to years within mammalian tissues. Protein lifespan is relevant to organismal aging, as long-lived proteins can accrue damage over time. It is unclear how protein lifetime is shaped by tissue context, where both cell division and proteolytic degradation contribute to protein turnover. Here, we develop turnover and replication analysis by 15N isotope labeling (TRAIL) to quantify both protein and cell lifetimes with high precision and no toxicity over a 32-day labeling period across 4 mammalian tissues. We report that cell division promotes non-selective protein turnover in proliferative tissues, while physicochemical features such as hydrophobicity, charge, and intrinsic disorder exert a significant influence on protein turnover only in non-proliferative tissues. Protein lifetimes vary non-randomly across tissues after correcting for differences in cell division rate. Multiprotein complexes such as the ribosome have highly consistent lifetimes across tissues, while mitochondria, peroxisomes, and lipid droplets have variable lifetimes. These data indicate that cell turnover, sequence-encoded features, and other environmental factors modulate protein lifespan in vivo. In the future, TRAIL can be used to explore how environment, aging, and disease affect tissue homeostasis.

systems biology↗

bioRxiv2019

Selective clearance of the inner nuclear membrane protein emerin by vesicular transport during ER stress

The inner nuclear membrane (INM) is a subdomain of the endoplasmic reticulum (ER) that is gated by the nuclear pore complex. It is unknown whether proteins of the INM and ER are degraded through shared or distinct pathways in mammalian cells. We applied dynamic proteomics to profile protein half-lives and report that INM and ER residents turn over at similar rates, indicating that the INMs unique topology is not a barrier to turnover. Using a microscopy approach, we observed that the proteasome can degrade INM proteins in situ. However, we also uncovered evidence for selective, vesicular transport-mediated turnover of a single INM protein, emerin, that is potentiated by ER stress. Emerin is rapidly cleared from the INM by a mechanism that requires emerins LEM domain to mediate vesicular trafficking to lysosomes. This work demonstrates that the INM can be dynamically remodeled in response to environmental inputs.

cell biology↗