Direct force probe reveals the mechanics of nuclear homeostasis in the mammalian cell

How cells maintain nuclear shape and position against various intracellular and extracellular forces is not well understood, although defects in nuclear mechanical homeostasis are associated with a variety of human diseases. We estimated the force required to displace and deform the nucleus in adherent living cells with a technique to locally pull the nuclear surface. A minimum pulling force of a few nanonewtons--far greater than typical intracellular motor forces--was required to significantly displace and deform the nucleus. Upon force removal, the original shape and position were restored quickly within a few seconds. This stiff, elastic response required the presence of vimentin, lamin A/C, and SUN (Sad1p, UNC-84)-domain protein linkages, but not F-actin or microtubules. Although F-actin and microtubules are known to exert mechanical forces on the nuclear surface through molecular motor activity, we conclude that the intermediate filament networks maintain nuclear mechanical homeostasis against localized forces

Functional association of Sun1 with nuclear pore complexes

Sun1 and 2 are A-type lamin-binding proteins that, in association with nesprins, form a link between the inner nuclear membranes (INMs) and outer nuclear membranes of mammalian nuclear envelopes. Both immunofluorescence and immunoelectron microscopy reveal that Sun1 but not Sun2 is intimately associated with nuclear pore complexes (NPCs). Topological analyses indicate that Sun1 is a type II integral protein of the INM. Localization of Sun1 to the INM is defined by at least two discrete regions within its nucleoplasmic domain. However, association with NPCs is dependent on the synergy of both nucleoplasmic and lumenal domains. Cells that are either depleted of Sun1 by RNA interference or that overexpress dominant-negative Sun1 fragments exhibit clustering of NPCs. The implication is that Sun1 represents an important determinant of NPC distribution across the nuclear surface

Characterization of a recurrent missense mutation in the forkhead DNA-binding domain of \u3ci\u3eFOXP1\u3c/i\u3e

Haploinsufficiency of Forkhead box protein P1 (FOXP1), a highly conserved transcription factor, leads to developmental delay, intellectual disability, autism spectrum disorder, speech delay, and dysmorphic features. Most of the reported FOXP1 mutations occur on the C-terminus of the protein and cluster around to the forkhead domain. All reported FOXP1 pathogenic variants result in abnormal cellular localization and loss of transcriptional repression activity of the protein product. Here we present three patients with the same FOXP1 mutation, c.1574G\u3eA (p.R525Q), that results in the characteristic loss of transcription repression activity. This mutation, however, represents the first reported FOXP1 mutation that does not result in cytoplasmic or nuclear aggregation of the protein but maintains normal nuclear localization

Identification of Redox and Glucose-Dependent Txnip Protein Interactions

Thioredoxin-interacting protein (Txnip) acts as a negative regulator of thioredoxin function and is a critical modulator of several diseases including, but not limited to, diabetes, ischemia-reperfusion cardiac injury, and carcinogenesis. Therefore, Txnip has become an attractive therapeutic target to alleviate disease pathologies. Although Txnip has been implicated with numerous cellular processes such as proliferation, fatty acid and glucose metabolism, inflammation, and apoptosis, the molecular mechanisms underlying these processes are largely unknown. The objective of these studies was to identify Txnip interacting proteins using the proximity-based labeling method, BioID, to understand differential regulation of pleiotropic Txnip cellular functions. The BioID transgene fused to Txnip expressed in HEK293 identified 31 interacting proteins. Many protein interactions were redox-dependent and were disrupted through mutation of a previously described reactive cysteine (C247S). Furthermore, we demonstrate that this model can be used to identify dynamic Txnip interactions due to known physiological regulators such as hyperglycemia. These data identify novel Txnip protein interactions and demonstrate dynamic interactions dependent on redox and glucose perturbations, providing clarification to the pleiotropic cellular functions of Txnip

Chromatin targeting of the RNF12/RLIM E3 ubiquitin ligase controls transcriptional responses

Protein ubiquitylation regulates key biological processes including transcription. This isexemplified by the E3 ubiquitin ligase RNF12/RLIM, which controls developmental geneexpression by ubiquitylating the REX1 transcription factor and is mutated in an X-linkedintellectual disability disorder. However, the precise mechanisms by which ubiquitylationdrives specific transcriptional responses are not known. Here, we show that RNF12 isrecruited to specific genomic locations via a consensus sequence motif, which enables colocalisationwith REX1 substrate at gene promoters. Surprisingly, RNF12 chromatinrecruitment is achieved via a non-catalytic basic region and comprises a previouslyunappreciated N-terminal autoinhibitory mechanism. Furthermore, RNF12 chromatintargeting is critical for REX1 ubiquitylation and downstream RNF12-dependent generegulation. Our results demonstrate a key role for chromatin in regulation of the RNF12-REX1axis and provide insight into mechanisms by which protein ubiquitylation enablesprogramming of gene expression

Heat shock protein Grp78/BiP/HspA5 binds directly to TDP-43 and mitigates toxicity associated with disease pathology

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with no cure or effective treatment in which TAR DNA Binding Protein of 43 kDa (TDP-43) abnormally accumulates into misfolded protein aggregates in affected neurons. It is widely accepted that protein misfolding and aggregation promotes proteotoxic stress. The molecular chaperones are a primary line of defense against proteotoxic stress, and there has been long-standing interest in understanding the relationship between chaperones and aggregated protein in ALS. Of particular interest are the heat shock protein of 70 kDa (Hsp70) family of chaperones. However, defining which of the 13 human Hsp70 isoforms is critical for ALS has presented many challenges. To gain insight into the specific Hsp70 that modulates TDP-43, we investigated the relationship between TDP-43 and the Hsp70s using proximity-dependent biotin identification (BioID) and discovered several Hsp70 isoforms associated with TDP-43 in the nucleus, raising the possibility of an interaction with native TDP-43. We further found that HspA5 bound specifically to the RNA-binding domain of TDP-43 using recombinantly expressed proteins. Moreover, in a Drosophila strain that mimics ALS upon TDP-43 expression, the mRNA levels of the HspA5 homologue (Hsc70.3) were significantly increased. Similarly we observed upregulation of HspA5 in prefrontal cortex neurons from human ALS patients. Finally, overexpression of HspA5 in Drosophila rescued TDP-43-induced toxicity, suggesting that upregulation of HspA5 may have a compensatory role in ALS pathobiology

The mammalian LINC complex regulates genome transcriptional responses to substrate rigidity

Mechanical integration of the nucleus with the extracellular matrix (ECM) is established by linkage between the cytoskeleton and the nucleus. This integration is hypothesized to mediate sensing of ECM rigidity, but parsing the function of nucleus-cytoskeleton linkage from other mechanisms has remained a central challenge. Here we took advantage of the fact that the LINC (linker of nucleoskeleton and cytoskeleton) complex is a known molecular linker of the nucleus to the cytoskeleton, and asked how it regulates the sensitivity of genome-wide transcription to substratum rigidity. We show that gene mechanosensitivity is preserved after LINC disruption, but reversed in direction. Combined with myosin inhibition studies, we identify genes that depend on nuclear tension for their regulation. We also show that LINC disruption does not attenuate nuclear shape sensitivity to substrate rigidity. Our results show for the first time that the LINC complex facilitates mechano-regulation of expression across the genome

Joint international consensus statement for ending stigma of obesity

People with obesity commonly face a pervasive, resilient form of social stigma. They are often subject to discrimination in the workplace as well as in educational and healthcare settings. Research indicates that weight stigma can cause physical and psychological harm, and that affected individuals are less likely to receive adequate care. For these reasons, weight stigma damages health, undermines human and social rights, and is unacceptable in modern societies. To inform healthcare professionals, policymakers, and the public about this issue, a multidisciplinary group of international experts, including representatives of scientific organizations, reviewed available evidence on the causes and harms of weight stigma and, using a modified Delphi process, developed a joint consensus statement with recommendations to eliminate weight bias. Academic institutions, professional organizations, media, public-health authorities, and governments should encourage education about weight stigma to facilitate a new public narrative about obesity, coherent with modern scientific knowledge

Human skeletal muscle is refractory to the anabolic effects of leucine during the postprandial muscle-full period in older men

Leucine modulates muscle protein synthesis (MPS), with potential to facilitate accrual/maintenance of muscle mass. Animal models suggest that leucine boluses shortly after meals may prolong MPS and delay onset of a “muscle-full” state. However, the effects of nutrient “top-ups” in humans, and particularly older adults where deficits exist, have not been explored. We determined the effects of a leucine top-up after essential amino acid (EAA) feeding on anabolic signaling, MPS, and muscle energy metabolism in older men. During 13C6-phenylalanine infusion, 16 men (∼70 years) consumed 15 g of EAA with (n=8, FED + LEU) or without (n=8, FED) 3 g of leucine top-up 90 min later. Repeated blood and muscle sampling permitted measurement of fasting and postprandial plasma EAA, insulin, anabolic signaling including mTOR complex 1 (mTORC1) substrates, cellular ATP and phosphorylocreatine, and MPS. Oral EAA achieved rapid insulinemia (12.5 iU·ml−1 25 min post-feed), essential aminoacidemia (3000 μM, 45–65 min post-feed), and activation of mTORC1 signaling. Leucine top-up prolonged plasma EAA (2800 μM, 135 min) and leucine availability (1050 μM, 135 min post-feed). Fasting FSRs of 0.046 and 0.056%·h-1 (FED and FED + LEU respectively) increased to 0.085 and 0.085%·h-1 90–180 min post-feed and returned to basal rates after 180 min in both groups. Phosphorylation of mTORC1 substrates returned to fasting levels 240 min post-feed in both groups. Feeding had limited effect on muscle elongation factor 2 (eEF2) phosphorylation. We demonstrate the refractoriness of muscle to nutrient-led anabolic stimulation in the postprandial period; thus, leucine supplements should be taken outside of meals, or with meals containing suboptimal protein in terms of either amount or EAA composition