LAG3 associates with TCR-CD3 complexes and suppresses signaling by driving co-receptor-Lck dissociation.

LAG3 is an inhibitory receptor that is highly expressed on exhausted T cells. Although LAG3-targeting immunotherapeutics are currently in clinical trials, how LAG3 inhibits T cell function remains unclear. Here, we show that LAG3 moved to the immunological synapse and associated with the T cell receptor (TCR)-CD3 complex in CD4 and CD8 T cells, in the absence of binding to major histocompatibility complex class II-its canonical ligand.

Cytoneme delivery of Sonic Hedgehog from ligand-producing cells requires Myosin 10 and a Dispatched-BOC/CDON co-receptor complex.

Morphogens function in concentration-dependent manners to instruct cell fate during tissue patterning. The cytoneme morphogen transport model posits that specialized filopodia extend between morphogen-sending and responding cells to ensure that appropriate signaling thresholds are achieved. How morphogens are transported along and deployed from cytonemes, how quickly a cytoneme-delivered, receptor-dependent signal is initiated, and whether these processes are conserved across phyla are not known.

MEKK3-MEK5-ERK5 signaling promotes mitochondrial degradation.

Mitochondria are vital organelles that coordinate cellular energy homeostasis and have important roles in cell death. Therefore, the removal of damaged or excessive mitochondria is critical for maintaining proper cellular function. The PINK1-Parkin pathway removes acutely damaged mitochondria through a well-characterized mitophagy pathway, but basal mitochondrial turnover occurs via distinct and less well-understood mechanisms.

Loss of MAGEL2 in Prader-Willi syndrome leads to decreased secretory granule and neuropeptide production.

Prader-Willi syndrome (PWS) is a developmental disorder caused by loss of maternally imprinted genes on 15q11-q13, including melanoma antigen gene family member L2 (MAGEL2). The clinical phenotypes of PWS suggest impaired hypothalamic neuroendocrine function; however, the exact cellular defects are unknown. Here, we report deficits in secretory granule (SG) abundance and bioactive neuropeptide production upon loss of MAGEL2 in humans and mice.

Chronic optogenetic induction of stress granules is cytotoxic and reveals the evolution of ALS-FTD pathology.

Unlabelled: Stress granules (SGs) are non-membrane-bound RNA-protein granules that assemble through phase separation in response to cellular stress. Disturbances in SG dynamics have been implicated as a primary driver of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), suggesting the hypothesis that these diseases reflect an underlying disturbance in the dynamics and material properties of SGs. However, this concept has remained largely untestable in available models of SG assembly, which require the confounding variable of exogenous stressors.

An unexpected protein interaction promotes drug resistance in leukemia.

The overall survival of patients with acute myeloid leukemia (AML) is poor and identification of new disease-related therapeutic targets remains a major goal for this disease. Here we show that expression of MPP1, a PDZ-domain-containing protein, highly correlated with ABCC4 in AML, is associated with worse overall survival in AML. Murine hematopoietic progenitor cells overexpressing MPP1 acquired the ability to serially replate in methylcellulose culture, a property crucially dependent upon ABCC4.

TIA1 Mutations in Amyotrophic Lateral Sclerosis and Frontotemporal Dementia Promote Phase Separation and Alter Stress Granule Dynamics.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are age-related neurodegenerative disorders with shared genetic etiologies and overlapping clinical and pathological features. Here we studied a novel ALS/FTD family and identified the P362L mutation in the low-complexity domain (LCD) of T cell-restricted intracellular antigen-1 (TIA1). Subsequent genetic association analyses showed an increased burden of TIA1 LCD mutations in ALS patients compared to controls (p = 8.

Drebrin-mediated microtubule-actomyosin coupling steers cerebellar granule neuron nucleokinesis and migration pathway selection.

Neuronal migration from a germinal zone to a final laminar position is essential for the morphogenesis of neuronal circuits. While it is hypothesized that microtubule-actomyosin crosstalk is required for a neuron's 'two-stroke' nucleokinesis cycle, the molecular mechanisms controlling such crosstalk are not defined. By using the drebrin microtubule-actin crosslinking protein as an entry point into the cerebellar granule neuron system in combination with super-resolution microscopy, we investigate how these cytoskeletal systems interface during migration.

C9orf72 Dipeptide Repeats Impair the Assembly, Dynamics, and Function of Membrane-Less Organelles.

Expansion of a hexanucleotide repeat GGGGCC (GC) in C9ORF72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Transcripts carrying (GC) expansions undergo unconventional, non-ATG-dependent translation, generating toxic dipeptide repeat (DPR) proteins thought to contribute to disease. Here, we identify the interactome of all DPRs and find that arginine-containing DPRs, polyGly-Arg (GR) and polyPro-Arg (PR), interact with RNA-binding proteins and proteins with low complexity sequence domains (LCDs) that often mediate the assembly of membrane-less organelles.

IRGB10 Liberates Bacterial Ligands for Sensing by the AIM2 and Caspase-11-NLRP3 Inflammasomes.

The inflammasome is an intracellular signaling complex, which on recognition of pathogens and physiological aberration, drives activation of caspase-1, pyroptosis, and the release of the pro-inflammatory cytokines IL-1β and IL-18. Bacterial ligands must secure entry into the cytoplasm to activate inflammasomes; however, the mechanisms by which concealed ligands are liberated in the cytoplasm have remained unclear. Here, we showed that the interferon-inducible protein IRGB10 is essential for activation of the DNA-sensing AIM2 inflammasome by Francisella novicida and contributed to the activation of the LPS-sensing caspase-11 and NLRP3 inflammasome by Gram-negative bacteria.

Phase separation by low complexity domains promotes stress granule assembly and drives pathological fibrillization.

Stress granules are membrane-less organelles composed of RNA-binding proteins (RBPs) and RNA. Functional impairment of stress granules has been implicated in amyotrophic lateral sclerosis, frontotemporal dementia, and multisystem proteinopathy-diseases that are characterized by fibrillar inclusions of RBPs. Genetic evidence suggests a link between persistent stress granules and the accumulation of pathological inclusions.

Outer Hair Cell Lateral Wall Structure Constrains the Mobility of Plasma Membrane Proteins.

Nature's fastest motors are the cochlear outer hair cells (OHCs). These sensory cells use a membrane protein, Slc26a5 (prestin), to generate mechanical force at high frequencies, which is essential for explaining the exquisite hearing sensitivity of mammalian ears. Previous studies suggest that Slc26a5 continuously diffuses within the membrane, but how can a freely moving motor protein effectively convey forces critical for hearing? To provide direct evidence in OHCs for freely moving Slc26a5 molecules, we created a knockin mouse where Slc26a5 is fused with YFP.

Cross-species genomic and epigenomic landscape of retinoblastoma.

Genetically engineered mouse models (GEMMs) of human cancer are important for advancing our understanding of tumor initiation and progression as well as for testing novel therapeutics. Retinoblastoma is a childhood cancer of the developing retina that initiates with biallelic inactivation of the RB1 gene. GEMMs faithfully recapitulate the histopathology, molecular, cellular, morphometric, neuroanatomical and neurochemical features of human retinoblastoma.

Distinct TCR signaling pathways drive proliferation and cytokine production in T cells.

The physiological basis and mechanistic requirements for a large number of functional immunoreceptor tyrosine-based activation motifs (ITAMs; high ITAM multiplicity) in the complex of the T cell antigen receptor (TCR) and the invariant signaling protein CD3 remain obscure. Here we found that whereas a low multiplicity of TCR-CD3 ITAMs was sufficient to engage canonical TCR-induced signaling events that led to cytokine secretion, a high multiplicity of TCR-CD3 ITAMs was required for TCR-driven proliferation. This was dependent on the formation of compact immunological synapses, interaction of the adaptor Vav1 with phosphorylated CD3 ITAMs to mediate the recruitment and activation of the oncogenic transcription factor Notch1 and, ultimately, proliferation induced by the cell-cycle regulator c-Myc.

Anti-apoptotic MCL-1 localizes to the mitochondrial matrix and couples mitochondrial fusion to respiration.

MCL-1, an anti-apoptotic BCL-2 family member that is essential for the survival of multiple cell lineages, is also among the most highly amplified genes in cancer. Although MCL-1 is known to oppose cell death, precisely how it functions to promote survival of normal and malignant cells is poorly understood. Here, we report that different forms of MCL-1 reside in distinct mitochondrial locations and exhibit separable functions.

A unified model of mammalian BCL-2 protein family interactions at the mitochondria.

During apoptosis, the BCL-2 protein family controls mitochondrial outer membrane permeabilization (MOMP), but the dynamics of this regulation remain controversial. We employed chimeric proteins composed of exogenous BH3 domains inserted into a tBID backbone that can activate the proapoptotic effectors BAX and BAK to permeabilize membranes without being universally sequestered by all antiapoptotic BCL-2 proteins. We thus identified two "modes" whereby prosurvival BCL-2 proteins can block MOMP, by sequestering direct-activator BH3-only proteins ("MODE 1") or by binding active BAX and BAK ("MODE 2").

Directed evolution of an extremely stable fluorescent protein.

In this paper we describe the evolution of eCGP123, an extremely stable green fluorescent protein based on a previously described fluorescent protein created by consensus engineering (CGP: consensus green protein). eCGP123 could not be denatured by a standard thermal melt, preserved almost full fluorescence after overnight incubation at 80 degrees C and possessed a free energy of denaturation of 12.4 kcal/mol.

Measuring an antibody affinity distribution molecule by molecule.

Single molecule fluorescence microscopy was used to observe the binding and unbinding of hapten decorated quantum dots to individual surface immobilized antibodies. The fluorescence time history from an individual antibody site can be used to calculate its binding affinity. While quantum dot blinking occurs during these measurements, we describe a simple empirical method to correct the apparent/observed affinity to account for the blinking contribution.

The creation of a novel fluorescent protein by guided consensus engineering.

Consensus engineering has been used to increase the stability of a number of different proteins, either by creating consensus proteins from scratch or by modifying existing proteins so that their sequences more closely match a consensus sequence. In this paper we describe the first application of consensus engineering to the ab initio creation of a novel fluorescent protein. This was based on the alignment of 31 fluorescent proteins with >62% homology to monomeric Azami green (mAG) protein, and used the sequence of mAG to guide amino acid selection at positions of ambiguity.