8 research outputs found

    Hydrogen/Deuterium Exchange Mass Spectrometry Provides Insights into the Role of Drosophila Testis-Specific Myosin VI Light Chain AndroCaM

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    In Drosophila testis, myosin VI plays a special role, distinct from its motor function, by anchoring components to the unusual actin-based structures (cones) that are required for spermatid individualization. For this, the two calmodulin (CaM) light-chain molecules of myosin VI are replaced by androcam (ACaM), a related protein with 67% identity to CaM. Although ACaM has a similar bi-lobed structure to CaM, with two EF hand-type Ca2+ binding sites per lobe, only one functional Ca2+ binding site operates in the amino-terminus. To understand this light chain substitution, we used hydrogen–deuterium exchange mass spectrometry (HDX-MS) to examine dynamic changes in ACaM and CaM upon Ca2+ binding and interaction with the two CaM binding motifs of myosin VI (insert2 and IQ motif). HDX-MS reveals that binding of Ca2+ to ACaM destabilizes its N-lobe but stabilizes the entire C-lobe, whereas for CaM, Ca2+ binding induces a pattern of alternating stabilization/destabilization throughout. The conformation of this stable holo-C-lobe of ACaM seems to be a “prefigured” version of the conformation adopted by the holo-C-lobe of CaM for binding to insert2 and the IQ motif of myosin VI. Strikingly, the interaction of holo-ACaM with either peptide converts the holo-N-lobe to its Ca2+-free, more stable, form. Thus, ACaM in vivo should bind the myosin VI light chain sites in an apo-N-lobe/holo-C-lobe state that cannot fulfill the Ca2+-related functions of holo-CaM required for myosin VI motor assembly and activity. These findings indicate that inhibition of myosin VI motor activity is a precondition for transition to an anchoring function

    Additional file 3: of Isoform-specific deletion of PKM2 constrains tumor initiation in a mouse model of soft tissue sarcoma

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    Figure S3. (A) Representative images of IF for tdTomato, PKM1 or PKM2, and EdU on KP M2 +/+ or KP M2 −/− sarcoma cell lines. (B) Cumulative populaion doublings of KP M2 +/+ or KP M2 −/− sarcoma cell lines, n = 3 KP M2 +/+ cell lines and n = 4 KP M2 −/− cell lines. (C) Sarcoma weight in grams. (JPG 2694 kb

    Additional file1: of Isoform-specific deletion of PKM2 constrains tumor initiation in a mouse model of soft tissue sarcoma

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    Figure S1. Quantification of PKM1 and PKM2 staining intensities shown as percent of tissue cores scored in 16 normal human skeletal muscle samples (A) and 48 primary human rhabdomyosarcomas (B). Score 0 = no staining, Score 1 = weak, Score 2 = positive, or Score 3 = strong. (C) Representative images of IHC for PKM1 and PKM2 in KP mouse sarcoma tissue. Corresponding H&E images are shown. Scale bars, 20 μm. (JPG 1314 kb

    Additional file 4: of Isoform-specific deletion of PKM2 constrains tumor initiation in a mouse model of soft tissue sarcoma

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    Table S1. Raw data for LC-MS metabolomics on KP M2+/+ and KP M2-/- sarcomas. Related to Fig. 5. This table contains all the LC-MS metabolomics data. Includes normalization and statistical analysis for each measured metabolite. (XLSX 140 kb
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