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News - Jul 23, 2014

Flanagan Lab finds a novel RNA-based mechanism for microtubule regulation
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Microtubules are fundamental for the spatial organization and motility of neurons and other cells.  In a recent article in Cell, the Flanagan Lab identified a novel function for the microtubule plus-end protein Adenomatous polyposis coli (APC), a scaffold protein known to be important in biology and disease. They found that APC is an RNA-binding protein, and identified an mRNA interactome, which was highly enriched for APC-related functions, including microtubule organization, cell motility, cancer and neurologic disease. Among the targets were tubulin mRNAs, and further studies showed that regulation of β2B-tubulin mRNA is critical for dynamic microtubule extension in axons, and for cortical neuron migration in vivo.  These results lead to a novel protein-synthesis-based model for microtubule assembly, and identify APC as a platform linking protein and mRNA networks in normal and disease states.

News - Jun 18, 2014

Gygi Lab develops new "viromics" technique to analyze host-pathogen interactions
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A systematic quantitative analysis of temporal changes in host and viral proteins throughout the course of a productive infection could provide dynamic insights into virus-host interaction. In a recent article in Cell, the Gygi Lab describes a novel proteomic technique, ‘quantitative temporal viromics’ (QTV), employing multiplexed tandem mass tag-based mass spectrometry. They apply this technology to human cytomegalovirus (HCMV), not only an important pathogen but a paradigm of viral immune evasion. QTV detailed how HCMV orchestrates the expression of >8,000 cellular proteins, including 1,200 cell surface proteins, to manipulate signaling pathways and counter intrinsic, innate, and adaptive immune defenses. QTV predicted novel natural killer and T-cell ligands, as well as 29 viral proteins present at the cell surface--potential new therapeutic targets. Temporal profiles of >80% of HCMV canonical genes and 14 non-canonical HCMV ORFs were defined. QTV is a powerful, novel method that can yield important insights into viral infection, and is applicable to any virus with a robust in vitro model.  This work was in collaboration with the Wilkinson Lab (Cardiff University, UK) and Lehner Lab (Cambridge University, UK).

Figure: schematic of QTV strategy, as applied to HCMV.

News - Jun 10, 2014

Reck-Peterson Lab illuminates how dynein gets to the start of its track
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Cytoplasmic dynein is the major motor protein that transports cargoes toward the minus end of microtubules. In most cell types, this corresponds to movement toward the cell interior. However, if dynein only moves to the minus end of microtubules, a problem arises: how would dynein initially reach the plus end of the microtubule and the outskirts of the cell, where it collects cargoes? In a recent eLIFE article, the Reck-Peterson Lab reveals that a group of three proteins can solve this problem by transporting dynein to the plus end of the microtubule. The proteins comprise a plus-end-directed kinesin motor, and two additional proteins that connect dynein to the kinesin. Imaging the transport process shows that the dynein motor is not a passive passenger: it is able to resist against the kinesin. However, an additional microtubule-associated protein can help the kinesin motor to win this “tug of war”, and so the protein complex—including the dynein motor—moves toward the plus end of the microtubule.

Figure: Kymograph showing a fluorescently labeled dynein construct (green) moving toward the plus end of the microtubule.

News - Jun 10, 2014

Pellman Lab finds that centrosome amplification may be advantageous for tumor progression
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In a recent article in Nature, the Pellman Lab reports that increased numbers of centrosomes can mimic and enhance the effects of a breast cancer oncogene. Centrosome amplification, a common cytoskeletal aberration in cancer cells, has long been recognized as a feature of human tumors, but its role in tumorigenesis has been unclear.  This amplification is poorly tolerated in non-transformed cells; extra centrosomes are spontaneously lost in the absence of selection. Thus, the high frequency of centrosome amplification, particularly in more aggressive tumors, presents something of a paradox and raised the possibility that extra centrosomes could confer advantageous characteristics that promote tumor progression.

Using a 3D model system and other approaches to culture human mammary epithelial cells, the Pellman Lab found that centrosome amplification triggers cell invasion. This invasive behavior is similar to that induced by overexpression of the breast cancer oncogene ERBB2 and enhances invasiveness triggered by high-level expression of ERBB2. Their data indicate that, through increased nucleation of microtubules from centrosomes, centrosome amplification increases the activity of the small GTPase, Rac1, disrupting normal cell-cell adhesion and promoting invasion. Activation of Rac is a well-established feature of cancer cells and is thought to be critical for cancer cell invasion and metastasis.  These findings demonstrate that centrosome amplification, a structural alteration of the cytoskeleton, can promote features of malignant transformation.

 

This work was in collaboration with the Thery Lab (Hospital Saint Louis), Brugge Lab (HMS, Cell Biology), and Polyak Lab (HMS & DFCI).  

The image illustrates the ability of centrosome amplification to promote invasive behavior of mammary epithelial cells.  Top image: normal, early stage  organoids in 3-D cultures; bottom image: organoids from cells with centrosome amplification. Laminin V is in green, actin is in red, and DNA is in blue.

News - Apr 30, 2014

Van Vactor Lab uncovers a novel regulatory mechanism in synaptogenesis

The formation of synaptic connections is a complex process involving the coordinated assembly of both pre- and post-synaptic compartments. Many evolutionarily conserved signaling pathways and gene networks have been shown to regulate synapse formation.  As recently described in Development, the Van Vactor Lab reported that the microRNA miR-8 modulates synapse structure by directly repressing the actin regulator Enabled (Ena) at the Drosophila neuromuscular junction (NMJ).  Localization to the area surrounding pre-synaptic boutons requires conserved motifs in the C-terminal actin-assembly domain of Ena-family proteins. Additional studies indicate that miR-8 controls NMJ architecture by inhibiting Ena expression in muscle and thus limiting post-synaptic actin assembly that would otherwise restrict the expansion of motor neuron terminals. This novel regulatory process coordinates the remodeling of pre- and post-synaptic compartments in NMJ morphogenesis.

Figure: Endogenous Ena (green) accumulates in the peribouton compartment with post-synaptic marker protein Discs large (red). 

News - Apr 04, 2014

Mechanism of ferritinophagy revealed by Harper Lab

Autophagy, the process by which proteins and organelles are sequestered in double-membrane structures called autophagosomes and delivered to lysosomes for degradation, is critical in diseases such as cancer and neurodegeneration, but our understanding of how cargo is selected and targeted to autophagosomes is incomplete. The Harper Lab in collaboration with Alec Kimmelman’s lab (DFCI) recently reported in Nature the use of quantitative proteomics to systematically identify autophagosome-enriched proteins, including cargo receptors. Among the novel autophagosomally enriched proteins was NCOA4, a cytoplasmic protein that they demonstrated to localize to autophagosomal vesicles in response to activation of autophagy. Unbiased identification of NCOA4-associated proteins revealed ferritin heavy and light chains, components of an iron-filled cage structure that protects cells from reactive iron species but is degraded via autophagy to release iron through an unknown mechanism. They found that delivery of ferritin to lysosomes required NCOA4, and an inability of NCOA4-deficient cells to degrade ferritin leads to decreased bioavailable intracellular iron. Their work identifies NCOA4 as a selective cargo receptor for autophagic turnover of ferritin (ferritinophagy) critical for iron homeostasis and provides a resource for further dissection of autophagosomal cargo-receptor connectivity.

Figure: Electron microscopy of purified autophagosomes employed for quantitative proteomics.

News - Mar 25, 2014

Danial Lab and collaborators identify a novel class of glucokinase activators

Glucokinase is a glucose-phosphorylating enzyme that regulates insulin release and hepatic metabolism, and its loss-of-function is implicated in the pathogenesis of diabetes. Glucokinase activators (GKAs) are attractive therapeutics in diabetes, however, clinical data indicate that their benefits can be offset by hypoglycemia due to marked allosteric enhancement of the enzyme’s glucose affinity. Recent collaborative studies between the Danial and Walensky laboratories led to the discovery of a novel class of GKAs. These findings were published in the January 2014 issue of Nature Structural & Molecular Biology and showed that the BCL-2 homology 3 (BH3) alpha-helix derived from human BAD, a glucokinase binding partner, increases the enzyme catalytic rate without dramatically changing glucose affinity. This provides a new mechanism for pharmacologic activation of glucokinase. Remarkably, BAD BH3 phospho-mimetic mediates these effects by engaging a novel region near the enzyme’s active site. This interaction increases insulin secretion in human islets and restores the function of naturally-occurring human glucokinase mutants at the active site. Thus, BAD phospho-mimetics may serve as a novel class of GKAs.

The figure demonstrates the interaction region of the BAD BH3 helix near the glucokinase active site where glucose binds. This site is distinct from the allosteric site of the enzyme shown in the reverse view.

News - Mar 25, 2014

Danial Lab explores the coordinate regulation of hepatic energy metabolism and gluconeogenesis by the dual-function protein BAD

The homeostatic balance of hepatic glucose utilization, storage and production is exquisitely controlled by hormonal signals and hepatic carbon metabolism during fed and fasted states. How the liver senses extracellular glucose to cue glucose utilization versus production is not fully understood. During short term fasting, glucose is produced by both net glycogenolysis and gluconeogenesis, whereas upon prolonged fasting, glucose is synthesized almost exclusively from gluconeogenesis. Abnormal elevation of hepatic glucose production is a chief determinant of fasting hyperglycemia in diabetes. In the February 2014 issue of Cell Metabolism, the Danial Lab reported that the physiologic balance of hepatic glycolysis and gluconeogenesis is regulated by BAD, a dual function protein with roles in apoptosis and metabolism. BAD deficiency reprograms hepatic substrate and energy metabolism towards diminished glycolysis, excess fatty acid oxidation and exaggerated glucose production that escapes suppression by insulin. The group conducted genetic and biochemical studies that revealed BAD’s suppression of gluconeogenesis is actuated by phosphorylation of its BH3 domain and subsequent activation of the glucose-phosphorylating enzyme glucokinase. They also found BAD-GK axis is required for suppression of hepatic glucose production by insulin. The physiologic relevance of these findings is evident from the ability of a BAD phospho-mimic variant to counteract unrestrained gluconeogenesis and improve glycemia in leptin resistant and high-fat diet models of diabetes and insulin resistance. These findings mark BAD as a regulator of hepatic substrate metabolism and insulin sensitivity.

News - Feb 28, 2014

Reck-Peterson Lab develops new screening methods to study intracellular transport

Eukaryotic cells transport macromolecules over long distances along the microtubule cytoskeleton using the molecular motors dynein and kinesin. One barrier to understanding how motors transport a vast array of cargos with spatial and temporal specificity is the lack of rapid genetic methods to identify new genes involved in the process. In the March 1 issue of Molecular Biology of the Cell, the Reck-Peterson Lab reported a microscopy-based screening method involving multiplexed genome sequencing in the model organism Aspergillus nidulans. A. nidulans, a filamentous fungus, is an ideal model to study transport because of its reliance on the microtubule cytoskeleton for growth, the ease of manipulating its genome using homologous recombination, and its well-characterized life cycle that is amenable to rapid genetic analysis. Using this new screening method, the lab discovered new alleles of motors and motor regulators. Both dynein and kinesin motors transport multiple cargos in A. nidulans. One of the major findings the lab made from studying new alleles of the dynein motor was that different dynein cargos have distinct requirements for motor speed, with some cargo (the nucleus) only requiring a dynein motor that can move at a fraction of its maximal speed, while another cargo (endosomes) requiring maximal dynein velocity. This screening method will likely pave the way for many additional discoveries about the mechanism and regulation of intracellular transport.

In the figure, nuclei distribute normally in wild-type A. nidulans hyphae (top), but not in hyphae lacking dynein (bottom). Slow-velocity dynein mutants can still distribute nuclei but not endosomes, revealing cargo-specific velocity requirements.

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