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Supplementary Legends 1-3 and Information from Sigma-1 Receptors Bind Cholesterol and Remodel Lipid Rafts in Breast Cancer Cell Lines

Lipid rafts are membrane platforms that spatially organize molecules for specific signaling pathways that regulate various cellular functions. Cholesterol is critical for liquid-ordered raft formation by serving as a spacer between the hydrocarbon chains of sphingolipids, and alterations in the cholesterol contents of the plasma membrane causes disruption of rafts. The role that σ receptors play in cancer is not clear, although it is frequently up-regulated in human cancer cells and tissues and σ receptors inhibit proliferation in carcinoma and melanoma cell lines, induce apoptosis in colon and mammary carcinoma cell lines, and reduce cellular adhesion in mammary carcinoma cell lines. In this study, we provide molecular and functional evidence for the involvement of the enigmatic σ1 receptors in lipid raft modeling by σ1 receptor–mediated cholesterol alteration of lipid rafts in breast cancer cell lines. Cholesterol binds to cholesterol recognition domains in the COOH terminus of the σ1 receptor. This binding is blocked by σ receptor drugs because the cholesterol-binding domains form part of the σ receptor drug-binding site, mutations of which abolish cholesterol binding. Furthermore, we outline a hypothetical functional model to explain the myriad of biological processes, including cancer, in which these mysterious receptors are involved. The findings of this study provide a biological basis for the potential therapeutic applications of lipid raft cholesterol regulation in cancer therapy using σ receptor drugs. [Cancer Res 2007;67(23):11166–75]

Cancer-causing missense mutations in the 3418 amino acid BRCA2 breast and ovarian cancer suppressor protein frequently affect a short (∼340 residue) segment in its carboxyl-terminal domain (DBD). Here, we identify a shared molecular mechanism underlying their pathogenicity. Pathogenic BRCA2 missense mutations cluster in the DBD’s helical domain (HD) and OB1-fold motifs, which engage the partner protein DSS1. Pathogenic - but not benign – DBD mutations weaken or abolish DSS1-BRCA2 assembly, provoking mutant BRCA2 oligomers that are excluded from the cell nucleus, and disable DNA repair by homologous DNA recombination (HDR). DSS1 inhibits the intracellular oligomerization of wildtype, but not mutant, forms of BRCA2. Remarkably, DSS1 expression corrects defective HDR in cells bearing pathogenic BRCA2 missense mutants with weakened, but not absent, DSS1 binding. Our findings identify a DSS1-mediated intracellular protein assembly mechanism that is disrupted by cancer-causing BRCA2 missense mutations, and suggest an approach for its therapeutic correction., Graphical Abstract Graphical AbstractCancer-causing mutations in the BRCA2 DBD impair DSS1 binding, provoking intracellular oligomerization and cytosolic mis-localization of the mutant BRCA2, and defective homologous recombination. These defects are reversed by DSS1 overexpression, for certain DBD mutants that retain small amounts of DSS1 binding.

Acknowledgements: I thank the Cambridge Institute for Medical Research flow cytometry core facility for cell sorting. Suzan Ber Esposito, Annie Howitt, Alessandro Esposito, and Andrew Fry kindly provided cDNA template plasmids. I thank Helen Pickersgill, Marco Sciacovelli, Dylan Ryan and Amy Emery for constructive comments on the draft manuscript., Funder: Medical Research Council; funder-id: http://dx.doi.org/10.13039/501100000265, Centrioles are non-membrane-bound organelles that participate in fundamental cellular processes through their ability to form physical contacts with other structures. During interphase, two mature centrioles can associate to form a single centrosome-a phenomenon known as centrosome cohesion. Centrosome cohesion is important for processes such as cell migration, and yet how it is maintained is unclear. Current models indicate that pericentriolar fibres termed rootlets, also known as the centrosome linker, entangle to maintain centriole proximity. Here, I uncover a centriole-centriole contact site and mechanism of centrosome cohesion based on coalescence of the proximal centriole component cNap1. Using live-cell imaging of endogenously tagged cNap1, I show that proximal centrioles form dynamic contacts in response to physical force from the cytoskeleton. Expansion microscopy reveals that cNap1 bridges between these contact sites, physically linking proximal centrioles on the nanoscale. Fluorescence correlation spectroscopy (FCS)-calibrated imaging shows that cNap1 accumulates at nearly micromolar concentrations on proximal centrioles, corresponding to a few hundred protein copy numbers. When ectopically tethered to organelles such as lysosomes, cNap1 forms viscous and cohesive assemblies that promote organelle spatial proximity. These results suggest a mechanism of centrosome cohesion by cNap1 at the proximal centriole and illustrate how a non-membrane-bound organelle forms organelle contact sites.

Life is spatially partitioned within lipid membranes to allow the isolated formation of distinct molecular states inside cells and organelles. Cell fusion is the merger of two or more cells to form a single cell. Here we provide a protocol for cell fusion of two different cell types. Fused hybrid cells are enriched by flow cytometry-based sorting, followed by fluorescence microscopy of hybrid cell structure and function. Fluorescently tagged proteins generated by genome editing are imaged inside fused cells, allowing cellular structures to be identified based on fluorescence emission and referenced back to the cell type of origin. This robust and general method can be applied to different cell types or organelles of interest, to understand cellular structure and function across a range of fundamental biological questions.

The human polo-like kinase PLK1 coordinates mitotic chromosome segregation by phosphorylating multiple chromatin- and kinetochore-binding proteins. How PLK1 activity is directed to specific substrates via phosphopeptide recognition by its carboxyl-terminal polo-box domain (PBD) is poorly understood. Here, we combine molecular, structural and chemical biology to identify a determinant for PLK1 substrate recognition that is essential for proper chromosome segregation. We show that mutations ablating an evolutionarily conserved, Tyr-lined pocket in human PLK1 PBD trigger cellular anomalies in mitotic progression and timing. Tyr pocket mutations selectively impair PLK1 binding to the kinetochore phosphoprotein substrate PBIP1, but not to the centrosomal substrate NEDD1. Through a structure-guided approach, we develop a small-molecule inhibitor, Polotyrin, which occupies the Tyr pocket. Polotyrin recapitulates the mitotic defects caused by mutations in the Tyr pocket, further evidencing its essential function, and exemplifying a new approach for selective PLK1 inhibition. Thus, our findings support a model wherein substrate discrimination via the Tyr pocket in the human PLK1 PBD regulates mitotic chromosome segregation to preserve genome integrity.

Despite widespread use the extent to which different mammalian transgene methods report on the properties of endogenous proteins has not been systematically compared. This study shows that the choice of fluorescence-tagging method fundamentally influences the ability to image the activity of the mitotic kinase Aurora B., Fluorescence tagging of proteins is a widely used tool to study protein function and dynamics in live cells. However, the extent to which different mammalian transgene methods faithfully report on the properties of endogenous proteins has not been studied comparatively. Here we use quantitative live-cell imaging and single-molecule spectroscopy to analyze how different transgene systems affect imaging of the functional properties of the mitotic kinase Aurora B. We show that the transgene method fundamentally influences level and variability of expression and can severely compromise the ability to report on endogenous binding and localization parameters, providing a guide for quantitative imaging studies in mammalian cells.

HsSAS-6 homodimers are present in the cytoplasm and assemble into ninefold symmetrical arrays at centrosomes, thus initiating procentriole formation., SAS-6 proteins are thought to impart the ninefold symmetry of centrioles, but the mechanisms by which their assembly occurs within cells remain elusive. In this paper, we provide evidence that the N-terminal, coiled-coil, and C-terminal domains of HsSAS-6 are each required for procentriole formation in human cells. Moreover, the coiled coil is necessary and sufficient to mediate HsSAS-6 centrosomal targeting. High-resolution imaging reveals that GFP-tagged HsSAS-6 variants localize in a torus around the base of the parental centriole before S phase, perhaps indicative of an initial loading platform. Moreover, fluorescence recovery after photobleaching analysis demonstrates that HsSAS-6 is immobilized progressively at centrosomes during cell cycle progression. Using fluorescence correlation spectroscopy and three-dimensional stochastic optical reconstruction microscopy, we uncover that HsSAS-6 is present in the cytoplasm primarily as a homodimer and that its oligomerization into a ninefold symmetrical ring occurs at centrioles. Together, our findings lead us to propose a mechanism whereby HsSAS-6 homodimers are targeted to centrosomes where the local environment and high concentration of HsSAS-6 promote oligomerization, thus initiating procentriole formation.

Understanding the component stoichiometry of the T cell antigen receptor (TCR) triggering apparatus is essential for building realistic models of signal initiation. Recent studies suggesting that the TCR and other signaling-associated proteins are preclustered on resting T cells relied on measurements of the behavior of membrane proteins at interfaces with functionalized glass surfaces. Using fluorescence recovery after photobleaching, we show that, compared with the apical surface, the mobility of TCRs is significantly reduced at Jurkat T cell/glass interfaces, in a signaling-sensitive manner. Using two biophysical approaches that mitigate these effects, bioluminescence resonance energy transfer and two-color coincidence detection microscopy, we show that, within the uncertainty of the methods, the membrane components of the TCR triggering apparatus, i.e. the TCR complex, MHC molecules, CD4/Lck and CD45, are exclusively monovalent or monomeric in human T cell lines, implying that TCR triggering depends only on the kinetics of TCR/pMHC interactions. These analyses also showed that constraining proteins to two dimensions at the cell surface greatly enhances random interactions versus those between the membrane and the cytoplasm. Simulations of TCR-pMHC complex formation based on these findings suggest how unclustered TCR triggering-associated proteins might nevertheless be capable of generating complex signaling outputs via the differential recruitment of cytosolic effectors to the cell membrane.

International audience; To prevent genome instability, mitotic exit is delayed until all chromosomes are properly attached to the mitotic spindle by the spindle assembly checkpoint (SAC). In this study, we characterized the function of ARHGEF17, identified in a genome-wide RNA interference screen for human mitosis genes. Through a series of quantitative imaging, biochemical, and biophysical experiments, we showed that ARHGEF17 is essential for SAC activity, because it is the major targeting factor that controls localization of the checkpoint kinase Mps1 to the kinetochore. This mitotic function is mediated by direct interaction of the central domain of ARHGEF17 with Mps1, which is autoregulated by the activity of Mps1 kinase, for which ARHGEF17 is a substrate. This mitosis-specific role is independent of ARHGEF17's RhoGEF activity in interphase. Our study thus assigns a new mitotic function to ARHGEF17 and reveals the molecular mechanism for a key step in SAC establishment.

Whether mitotic structures like the centrosome can self-organize from the regulated mobility of their dynamic protein components remains unclear. Here, we combine fluorescence spectroscopy and chemical genetics to study in living cells the diffusion of polo-like kinase 1 (PLK1), an enzyme critical for centrosome maturation at the onset of mitosis. The cytoplasmic diffusion of a functional EGFP-PLK1 fusion correlates inversely with known changes in its enzymatic activity during the cell cycle. Specific EGFP-PLK1 inhibition using chemical genetics enhances mobility, as do point mutations inactivating the polo-box or kinase domains responsible for substrate recognition and catalysis. Spatial mapping of EGFP-PLK1 diffusion across living cells, using raster image correlation spectroscopy and line scanning, detects regions of low mobility in centrosomes. These regions exhibit characteristics of increased transient recursive EGFP-PLK1 binding, distinct from the diffusion of stable EGFP-PLK1–containing complexes in the cytoplasm. Chemical genetic suppression of mitotic EGFP-PLK1 activity, even after centrosome maturation, causes defects in centrosome structure, which recover when activity is restored. Our findings imply that continuous PLK1 activity during mitosis maintains centrosome self-organization by a mechanism dependent on its reaction and diffusion, suggesting a model for the formation of stable mitotic structures using dynamic protein kinases.

Lipid rafts are membrane platforms that spatially organize molecules for specific signaling pathways that regulate various cellular functions. Cholesterol is critical for liquid-ordered raft formation by serving as a spacer between the hydrocarbon chains of sphingolipids, and alterations in the cholesterol contents of the plasma membrane causes disruption of rafts. The role that σ receptors play in cancer is not clear, although it is frequently up-regulated in human cancer cells and tissues and σ receptors inhibit proliferation in carcinoma and melanoma cell lines, induce apoptosis in colon and mammary carcinoma cell lines, and reduce cellular adhesion in mammary carcinoma cell lines. In this study, we provide molecular and functional evidence for the involvement of the enigmatic σ1 receptors in lipid raft modeling by σ1 receptor–mediated cholesterol alteration of lipid rafts in breast cancer cell lines. Cholesterol binds to cholesterol recognition domains in the COOH terminus of the σ1 receptor. This binding is blocked by σ receptor drugs because the cholesterol-binding domains form part of the σ receptor drug-binding site, mutations of which abolish cholesterol binding. Furthermore, we outline a hypothetical functional model to explain the myriad of biological processes, including cancer, in which these mysterious receptors are involved. The findings of this study provide a biological basis for the potential therapeutic applications of lipid raft cholesterol regulation in cancer therapy using σ receptor drugs. [Cancer Res 2007;67(23):11166–75]

To understand the function of cellular protein networks, spatial and temporal context is essential. Fluorescence correlation spectroscopy (FCS) is a single-molecule method to study the abundance, mobility and interactions of fluorescence-labeled biomolecules in living cells. However, manual acquisition and analysis procedures have restricted live-cell FCS to short-term experiments of a few proteins. Here, we present high-throughput (HT)-FCS, which automates screening and time-lapse acquisition of FCS data at specific subcellular locations and subsequent data analysis. We demonstrate its utility by studying the dynamics of 53 nuclear proteins. We made 60,000 measurements in 10,000 living human cells, to obtain biophysical parameters that allowed us to classify proteins according to their chromatin binding and complex formation. We also analyzed the cell-cycle-dependent dynamics of the mitotic kinase complex Aurora B/INCENP and showed how a rise in Aurora concentration triggers two-step complex formation. We expect that throughput and robustness will make HT-FCS a broadly applicable technology for characterizing protein network dynamics in cells.

A-type lamins encoded by LMNA form a structural fibrillar meshwork within the mammalian nucleus. How this nuclear organization may influence the execution of biological processes involving DNA transactions remains unclear. Here, we characterize changes in the dynamics and biochemical interactions of lamin A/C after DNA damage. We find that DNA breakage reduces the mobility of nucleoplasmic GFP-lamin A throughout the nucleus as measured by dynamic fluorescence imaging and spectroscopy in living cells, suggestive of incorporation into stable macromolecular complexes, but does not induce the focal accumulation of GFP-lamin A at damage sites. Using a proximity ligation assay and biochemical analyses, we show that lamin A engages chromatin via histone H2AX and its phosphorylated form (γH2AX) induced by DNA damage, and that these interactions are enhanced after DNA damage. Finally, we use three-dimensional time-lapse imaging to show that LMNA inactivation significantly reduces the positional stability of DNA repair foci in living cells. This defect is partially rescued by the stable expression of GFP-lamin A. Thus collectively, our findings suggest that the dynamic structural meshwork formed by A-type lamins anchors sites of DNA repair in mammalian nuclei, providing fresh insight into the control of DNA transactions by nuclear structural organization.

The DNA damage response encompasses a complex series of signaling pathways that function to regulate and facilitate the repair of damaged DNA. Recent studies have shown that the repair of transcriptionally inactive chromatin, named heterochromatin, is dependent upon the phosphorylation of the co-repressor, Krüppel-associated box (KRAB) domain-associated protein (KAP-1), by the ataxia telangiectasia-mutated (ATM) kinase. Co-repressors, such as KAP-1, function to regulate the rigid structure of heterochromatin by recruiting histone-modifying enzymes, such HDAC1/2, SETDB1, and nucleosome-remodeling complexes such as CHD3. Here, we have characterized a phosphorylation site in the HP1-binding domain of KAP-1, Ser-473, which is phosphorylated by the cell cycle checkpoint kinase Chk2. Expression of a nonphosphorylatable S473A mutant conferred cellular sensitivity to DNA-damaging agents and led to defective repair of DNA double-strand breaks in heterochromatin. In addition, cells expressing S473A also displayed defective mobilization of the HP1-β chromodomain protein. The DNA repair defect observed in cells expressing S473A was alleviated by depletion of HP1-β, suggesting that phosphorylation of KAP-1 on Ser-473 promotes the mobilization of HP1-β from heterochromatin and subsequent DNA repair. These results suggest a novel mechanism of KAP-1-mediated chromatin restructuring via Chk2-regulated HP1-β exchange from heterochromatin, promoting DNA repair.

A striking but poorly explained feature of cell division is the ability to assemble and maintain organelles not bounded by membranes, from freely diffusing components in the cytosol. This process is driven by information transfer across biological scales such that interactions at the molecular scale allow pattern formation at the scale of the organelle. One important example of such an organelle is the centrosome, which is the main microtubule organising centre in the cell. Centrosomes consist of two centrioles surrounded by a cloud of proteins termed the pericentriolar material (PCM). Profound structural and proteomic transitions occur in the centrosome during specific cell cycle stages, underlying events such as centrosome maturation during mitosis, in which the PCM increases in size and microtubule nucleating capacity. Here we use recent insights into the spatio-temporal behaviour of key regulators of centrosomal maturation, including Polo-like kinase 1, CDK5RAP2 and Aurora-A, to propose a model for the assembly and maintenance of the PCM through the mobility and local interactions of its constituent proteins. We argue that PCM structure emerges as a pattern from decentralised self-organisation through a reaction-diffusion mechanism, with or without an underlying template, rather than being assembled from a central structural template alone. Self-organisation of this kind may have broad implications for the maintenance of mitotic structures, which, like the centrosome, exist stably as supramolecular assemblies on the micron scale, based on molecular interactions at the nanometer scale.

The viral RNA (vRNA) genome of influenza A virus is replicated in the nucleus, exported to the cytoplasm as ribonucleoproteins (RNPs), and trafficked to the plasma membrane through uncertain means. Using fluorescent in situ hybridization to detect vRNA as well as the live cell imaging of fluorescently labeled RNPs, we show that an early event in vRNA cytoplasmic trafficking involves accumulation near the microtubule organizing center in multiple cell types and viral strains. Here, RNPs colocalized with Rab11, a pericentriolar recycling endosome marker. Cytoplasmic RNP localization was perturbed by inhibitors of vesicular trafficking, microtubules, or the short interfering RNA-mediated depletion of Rab11. Green fluorescent protein (GFP)-tagged RNPs in living cells demonstrated rapid, bidirectional, and saltatory movement, which is characteristic of microtubule-based transport, and also cotrafficked with fluorescent Rab11. Coprecipitation experiments showed an interaction between RNPs and the GTP-bound form of Rab11, potentially mediated via the PB2 subunit of the polymerase. We propose that influenza virus RNPs are routed from the nucleus to the pericentriolar recycling endosome (RE), where they access a Rab11-dependent vesicular transport pathway to the cell periphery.

How the biochemical reactions that lead to the repair of DNA damage are controlled by the diffusion and availability of protein reactants within the nucleoplasm is poorly understood. Here, we use gene targeting to replace Brca2 (a cancer suppressor protein essential for DNA repair) with a functional enhanced green fluorescent protein (EGFP)-tagged form, followed by fluorescence correlation spectroscopy to measure Brca2-EGFP diffusion in the nucleoplasm of living cells exposed to DNA breakage. Before damage, nucleoplasmic Brca2 molecules exhibit complex states of mobility, with long dwell times within a sub-fL observation volume, indicative of restricted motion. DNA damage significantly enhances the mobility of Brca2 molecules in the S/G2 phases of the cell cycle, via signaling through damage-activated protein kinases. Brca2 mobilization is accompanied by increased binding within the nucleoplasm to its cargo, the Rad51 recombinase, measured by fluorescence cross-correlation spectroscopy. Together, these results suggest that DNA breakage triggers the redistribution of soluble nucleoplasmic Brca2 molecules from a state of restricted diffusion, into a mobile fraction available for Rad51 binding. Our findings identify signal-regulated changes in nucleoplasmic protein diffusion as a means to control biochemical reactions in the cell nucleus.

Summary Background The Rad51 recombinase assembles on DNA to execute homologous DNA recombination (HR). This process is essential to repair replication-associated genomic lesions before cells enter mitosis, but how it is started and stopped during the cell cycle remains poorly understood. Rad51 assembly is regulated by the breast cancer suppressor Brca2, via its evolutionarily conserved BRC repeats, and a distinct carboxy (C)-terminal motif whose biological function is uncertain. Using "hit-and-run" gene targeting to insert single-codon substitutions into the avian Brca2 locus, we report here a previously unrecognized role for the C-terminal motif. Results We show that the avian C-terminal motif is functionally cognate with its human counterpart and identify point mutations that either abolish or enhance Rad51 binding. When these mutations are introduced into Brca2 , we find that they affect neither the assembly of Rad51 into nuclear foci on damaged DNA nor DNA repair by HR. Instead, foci disassemble more rapidly in a point mutant that fails to bind Rad51, associated with faster mitotic entry. Conversely, the slower disassembly of foci in a point mutant that constitutively binds Rad51 correlates with delayed mitosis. Indeed, Rad51 foci do not persist in mitotic cells even after G2 checkpoint suppression, suggesting that their disassembly is a prerequisite for chromosome segregation. Conclusions We conclude that Rad51 binding by the C-terminal Brca2 motif is dispensable for the execution of HR but instead links the disassembly of Rad51 complexes to mitotic entry. This mechanism may ensure that HR terminates before chromosome segregation. Our findings assign a biological function for the C-terminal Brca2 motif in a mechanism that coordinates DNA repair with the cell cycle.