Protocol for cytoskeleton staining of the semi-adherent multiple myeloma cell line RPMI 8226 by immunofluorescence.

Preservation of fine cellular details of semi-adherent or suspension cells for imaging by immunofluorescence is challenging. This protocol enables staining of floating cells with minimal morphological distortions, as we demonstrate with the semi-adherent multiple myeloma cell line RPMI 8226. We describe steps to better preserve structural details by fixing, permeabilizing, and staining cells in solution, while minimizing the number of centrifugation steps and centrifugation g-force. For complete details on the use and execution of this protocol, please refer to Osei-Amponsa et al.1.

hRpn13 shapes the proteome and transcriptome through epigenetic factors HDAC8, PADI4, and transcription factor NF-κB p50.

The anti-cancer target hRpn13 is a proteasome substrate receptor. However, hRpn13-targeting molecules do not impair its interaction with proteasomes or ubiquitin, suggesting other critical cellular activities. We find that hRpn13 depletion causes correlated proteomic and transcriptomic changes, with pronounced effects in myeloma cells for cytoskeletal and immune response proteins and bone-marrow-specific arginine deiminase PADI4. Moreover, a PROTAC against hRpn13 co-depletes PADI4, histone deacetylase HDAC8, and DNA methyltransferase MGMT. PADI4 binds and citrullinates hRpn13 and proteasomes, and proteasomes from PADI4-inhibited myeloma cells exhibit reduced peptidase activity. When off proteasomes, hRpn13 can bind HDAC8, and this interaction inhibits HDAC8 activity. Further linking hRpn13 to transcription, its loss reduces nuclear factor κB (NF-κB) transcription factor p50, which proteasomes generate by cleaving its precursor protein. NF-κB inhibition depletes hRpn13 interactors PADI4 and HDAC8. Altogether, we find that hRpn13 acts dually in protein degradation and expression and that proteasome constituency and, in turn, regulation varies by cell type.

A structurally conserved site in AUP1 binds the E2 enzyme UBE2G2 and is essential for ER-associated degradation.

Endoplasmic reticulum-associated degradation (ERAD) is a protein quality control pathway of fundamental importance to cellular homeostasis. Although multiple ERAD pathways exist for targeting topologically distinct substrates, all pathways require substrate ubiquitination. Here, we characterize a key role for the UBE2G2 Binding Region (G2BR) of the ERAD accessory protein ancient ubiquitous protein 1 (AUP1) in ERAD pathways. This 27-amino acid (aa) region of AUP1 binds with high specificity and low nanomolar affinity to the backside of the ERAD ubiquitin-conjugating enzyme (E2) UBE2G2. The structure of the AUP1 G2BR (G2BRAUP1) in complex with UBE2G2 reveals an interface that includes a network of salt bridges, hydrogen bonds, and hydrophobic interactions essential for AUP1 function in cells. The G2BRAUP1 shares significant structural conservation with the G2BR found in the E3 ubiquitin ligase gp78 and in vitro can similarly allosterically activate ubiquitination in conjunction with ERAD E3s. In cells, AUP1 is uniquely required to maintain normal levels of UBE2G2; this is due to G2BRAUP1 binding to the E2 and preventing its rapid degradation. In addition, the G2BRAUP1 is required for both ER membrane recruitment of UBE2G2 and for its activation at the ER membrane. Thus, by binding to the backside of a critical ERAD E2, G2BRAUP1 plays multiple critical roles in ERAD.

A computational model for classification of BRCA2 variants using mouse embryonic stem cell-based functional assays.

Sequencing-based genetic tests to identify individuals at increased risk of hereditary breast and ovarian cancers have resulted in the identification of more than 40,000 sequence variants of BRCA1 and BRCA2. A majority of these variants are considered to be variants of uncertain significance (VUS) because their impact on disease risk remains unknown, largely due to lack of sufficient familial linkage and epidemiological data. Several assays have been developed to examine the effect of VUS on protein function, which can be used to assess their impact on cancer susceptibility. In this study, we report the functional characterization of 88 BRCA2 variants, including several previously uncharacterized variants, using a well-established mouse embryonic stem cell (mESC)-based assay. We have examined their ability to rescue the lethality of Brca2 null mESC as well as sensitivity to six DNA damaging agents including ionizing radiation and a PARP inhibitor. We have also examined the impact of BRCA2 variants on splicing. In addition, we have developed a computational model to determine the probability of impact on function of the variants that can be used for risk assessment. In contrast to the previous VarCall models that are based on a single functional assay, we have developed a new platform to analyze the data from multiple functional assays separately and in combination. We have validated our VarCall models using 12 known pathogenic and 10 neutral variants and demonstrated their usefulness in determining the pathogenicity of BRCA2 variants that are listed as VUS or as variants with conflicting functional interpretation.

Fgf4 maintains Hes7 levels critical for normal somite segmentation clock function.

During vertebrate development, the presomitic mesoderm (PSM) periodically segments into somites, which will form the segmented vertebral column and associated muscle, connective tissue, and dermis. The periodicity of somitogenesis is regulated by a segmentation clock of oscillating Notch activity. Here, we examined mouse mutants lacking only Fgf4 or Fgf8, which we previously demonstrated act redundantly to prevent PSM differentiation. Fgf8 is not required for somitogenesis, but Fgf4 mutants display a range of vertebral defects. We analyzed Fgf4 mutants by quantifying mRNAs fluorescently labeled by hybridization chain reaction within Imaris-based volumetric tissue subsets. These data indicate that FGF4 maintains Hes7 levels and normal oscillatory patterns. To support our hypothesis that FGF4 regulates somitogenesis through Hes7, we demonstrate genetic synergy between Hes7 and Fgf4, but not with Fgf8. Our data indicate that Fgf4 is potentially important in a spectrum of human Segmentation Defects of the Vertebrae caused by defective Notch oscillations.

Investigation of F-BAR domain PACSIN proteins uncovers membrane tubulation function in cilia assembly and transport.

The intracellular ciliogenesis pathway requires membrane trafficking, fusion, and reorganization. Here, we demonstrate in human cells and zebrafish that the F-BAR domain containing proteins PACSIN1 and -2 play an essential role in ciliogenesis, similar to their binding partner and membrane reorganizer EHD1. In mature cilia, PACSINs and EHDs are dynamically localized to the ciliary pocket membrane (CPM) and transported away from this structure on membrane tubules along with proteins that exit the cilium. PACSINs function early in ciliogenesis at the ciliary vesicle (CV) stage to promote mother centriole to basal body transition. Remarkably, we show that PACSIN1 and EHD1 assemble membrane t7ubules from the developing intracellular cilium that attach to the plasma membrane, creating an extracellular membrane channel (EMC) to the outside of the cell. Together, our work uncovers a function for F-BAR proteins and membrane tubulation in ciliogenesis and explains how the intracellular cilium emerges from the cell.

Id1 Ablation Protects Hematopoietic Stem Cells from Stress-Induced Exhaustion and Aging.

Defining mechanisms that maintain tissue stem cells during homeostasis, stress, and aging is important for improving tissue regeneration and repair and enhancing cancer therapies. Here, we show that Id1 is induced in hematopoietic stem cells (HSCs) by cytokines that promote HSC proliferation and differentiation, suggesting that it functions in stress hematopoiesis. Genetic ablation of Id1 increases HSC self-renewal in serial bone marrow transplantation (BMT) assays, correlating with decreases in HSC proliferation, mitochondrial biogenesis, and reactive oxygen species (ROS) production. Id1-/- HSCs have a quiescent molecular signature and harbor less DNA damage than control HSCs. Cytokines produced in the hematopoietic microenvironment after γ-irradiation induce Id1 expression. Id1-/- HSCs display a blunted proliferative response to such cytokines and other inducers of chronic proliferation including genotoxic and inflammatory stress and aging, protecting them from chronic stress and exhaustion. Thus, targeting Id1 may be therapeutically useful for improving HSC survival and function during BMT, chronic stress, and aging.

BRCA2 minor transcript lacking exons 4-7 supports viability in mice and may account for survival of humans with a pathogenic biallelic mutation.

The breast cancer gene, BRCA2, is essential for viability, yet patients with Fanconi anemia-D1 subtype are born alive with biallelic mutations in this gene. The hypomorphic nature of the mutations is believed to support viability, but this is not always apparent. One such mutation is IVS7+2T>G, which causes premature protein truncation due to skipping of exon 7. We previously identified a transcript lacking exons 4-7, which restores the open-reading frame, encodes a DNA repair proficient protein and is expressed in IVS7+2T>G carriers. However, because the exons 4-7 encoded region contains several residues required for normal cell-cycle regulation and cytokinesis, this transcript's ability to support viability can be argued. To address this, we generated a Brca2 knock-in mouse model lacking exons 4-7 and demonstrated that these exons are dispensable for viability as well as tumor-free survival. This study provides the first in vivo evidence of the functional significance of a minor transcript of BRCA2 that can play a major role in the survival of humans who are homozygous for a clearly pathogenic mutation. Our results highlight the importance of assessing protein function restoration by premature truncating codon bypass by alternative splicing when evaluating the functional significance of variants such as nonsense and frame-shift mutations that are assumed to be clearly pathogenic. Our findings will impact not only the assessment of variants that map to this region, but also influence counseling paradigms and treatment options for such mutation carriers.

Plk1 relieves centriole block to reduplication by promoting daughter centriole maturation.

Centrosome overduplication promotes mitotic abnormalities, invasion and tumorigenesis. Cells regulate the number of centrosomes by limiting centriole duplication to once per cell cycle. The orthogonal orientation between a mother and a daughter centriole, established at the time of centriole duplication, is thought to block further duplication of the mother centriole. Loss of orthogonal orientation (disengagement) between two centrioles during anaphase is considered a licensing event for the next round of centriole duplication. Disengagement requires the activity of Polo-like kinase 1 (Plk1), but how Plk1 drives this process is not clear. Here we employ correlative live/electron microscopy and demonstrate that Plk1 induces maturation and distancing of the daughter centriole, allowing reduplication of the mother centriole even if the original daughter centriole is still orthogonal to it. We find that mother centrioles can undergo reduplication when original daughter centrioles are only ∼80 nm apart, which is the distance centrioles normally reach during prophase.

Pushing forces drive the comet-like motility of microtubule arrays in Dictyostelium.

Overexpression of dynein fragments in Dictyostelium induces the movement of the entire interphase microtubule array. Centrosomes in these cells circulate through the cytoplasm at rates between 0.4 and 2.5 microm/s and are trailed by a comet-tail like arrangement of the microtubule array. Previous work suggested that these cells use a dynein-mediated pulling mechanism to generate this dramatic movement and that similar forces are at work to maintain the interphase MTOC position in wild-type cells. In the present study, we address the nature of the forces used to produce microtubule movement. We have used a laser microbeam to sever the connection between the motile centrosomes and trailing microtubules, demonstrating that the major force for such motility results from a pushing on the microtubules. We eliminate the possibility that microtubule assembly/disassembly reactions are significant contributors to this motility and suggest that the cell cortex figures prominently in locating force-producing molecules. Our findings indicate that interphase microtubules in Dictyostelium are subject to both dynein- and kinesin-like forces and that these act in concert to maintain centrosome position in the cell and to support the radial character of the microtubule network.