Biology subjects

Hashimoto, H.

Publications and source records attributed to Hashimoto, H..

23 records · Page 2Linked to original sources

bioRxiv2020

Trans-scale-scope to find rare cellular activity in sub-million cells

In many phenomena of biological systems, not a majority, but a minority of cells act on the entire multicellular system causing drastic changes in the system properties. To understand the mechanisms underlying such phenomena, it is essential to observe the spatiotemporal dynamics of a huge population of cells at sub-cellular resolution, which is difficult with conventional tools such as microscopy and flow cytometry. Here, we describe an imaging system named AMATERAS that enables optical imaging with an over-one-centimeter field-of-view and a-few-micrometer spatial resolution. This trans-scale-scope has a simple configuration, composed of a low-power lens for machine vision and a hundred-megapixel image sensor. We demonstrated its high cell-throughput, capable of simultaneously observing more than one million cells. We applied it to dynamic imaging of calcium ions in HeLa cells and cyclic-adenosine-monophosphate in Dictyostelium discoideum, and successfully detected less than 0.01% of rare cells and observed multicellular events induced by these cells.

cell biology↗

bioRxiv2019

Structural Basis of Protein Arginine Methyltransferase Activation by a Catalytically Dead Homolog (Prozyme)

Prozymes are pseudoenzymes that stimulate the function of weakly active enzymes through complex formation. The major Trypanosoma brucei protein arginine methyltransferase, TbPRMT1 enzyme (ENZ), requires TbPRMT1 prozyme (PRO) to form an active heterotetrameric complex. Here we present the x-ray crystal structure of the ENZ-{Delta}52PRO tetrameric complex with the cofactor product S-adenosyl-L-homocysteine (AdoHcy) at 2.4 [A] resolution. The individual ENZ and PRO units adopt the highly-conserved PRMT domain architecture and form an antiparallel heterodimer that corresponds to the canonical homodimer observed in all previously reported PRMTs. In turn, two such heterodimers assemble into a tetramer both in the crystal and in solution with twofold rotational symmetry. ENZ is unstable in absence of PRO and incapable of forming a homodimer due to a steric clash of a non-conserved tyrosine within the dimerization arm, rationalizing why PRO is required to complement ENZ to form a PRMT dimer that is necessary, but not sufficient for PRMT activity. The PRO structure deviates from other, active PRTMs in that it lacks the conserved {eta}2 310-helix within the Rossmann fold, abolishing co-factor binding. In addition to its chaperone function for ENZ, PRO substantially contributes to substrate binding. Heterotetramerization is required for catalysis, since heterodimeric ENZ-PRO mutants lack binding affinity and methyltransferase activity towards the substrate protein TbRGG1. Together, we provide a structural basis for PRMT1 ENZ activation by PRO heterotetramer formation, which is conserved across all kinetoplastids, and describe a chaperone function of the PRMT1 prozyme, which represents a novel mode of PRMT regulation.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC=\"FIGDIR/small/776112v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (56K):\norg.highwire.dtl.DTLVardef@3d970dorg.highwire.dtl.DTLVardef@187f03forg.highwire.dtl.DTLVardef@1adee39org.highwire.dtl.DTLVardef@1f30f34_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG HighlightsO_LIThe crystal structure of Trypanosoma brucei protein arginine methyltransferase 1 (PRMT1) is reported\nC_LIO_LITwo enzyme-prozyme heterodimers form a stable heterotetramer essential for catalysis\nC_LIO_LIThe catalytically dead prozyme lacks elements essential for AdoMet binding\nC_LIO_LIThe enzyme alone is unstable and cannot form a canonical dimer due to steric clashes\nC_LIO_LIT. brucei PRMT1 prozyme adopts a chaperone function conserved across kinetoplastids\nC_LI

biochemistry↗

bioRxiv2019

Substrate Affinity and Specificity of the ScSth1p Bromodomain Are Fine-Tuned for Versatile Histone Recognition

Bromodomains recognize a wide range of acetylated lysine residues in histones and other nuclear proteins. Substrate specificity is critical for their biological function and arises from unique acetyl-lysine binding sites formed by variable loop regions. Here, we analyzed substrate affinity and specificity of the yeast ScSth1p bromodomain, an essential component of the "Remodels the Structure of Chromatin" complex, and found that the wild-type bromodomain preferentially recognizes H3K14ac and H4K20ac peptides. Mutagenesis studies--guided by our crystal structure determined at 2.7 [A] resolution--revealed loop residues Ser1276 and Trp1338 as key determinants for such interactions. Strikingly, point mutations of each of these residues substantially increased peptide binding affinity and selectivity, respectively. Our data demonstrate that the ScSth1p bromodomain is not optimized for binding to an individual acetylation mark, but fine-tuned for interactions with several such modifications, consistent with the versatile and multivalent nature of histone recognition by reader modules such as bromodomains. HighlightsO_LIThe ScSth1p bromodomain preferentially recognizes H3K14ac and H4K20ac peptides C_LIO_LISer1276 and Trp1338 are key determinants of substrate affinity and specificity C_LIO_LIMutations of these residues drastically increase substrate affinity and specificity C_LIO_LIThe ScSth1p bromodomain is fine-tuned for promiscuous histone tail recognition C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/559005v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@10c54b3org.highwire.dtl.DTLVardef@8cb555org.highwire.dtl.DTLVardef@1d4ef1eorg.highwire.dtl.DTLVardef@9eaf00_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

molecular biology↗

bioRxiv2018

Structural basis of HEAT-kleisin interactions in the human condensin I subcomplex

ABSTRUCTCondensin I is a multi-protein complex that plays an essential role in mitotic chromosome assembly and segregation in eukaryotes. It is composed of five subunits: two SMC (SMC2 and SMC4), a kleisin (CAP-H) and two HEAT-repeat (CAP-D2 and -G) subunits. Although it has been shown that balancing acts of the two HEAT-repeat subunits enable this complex to support dynamic assembly of chromosomal axes in vertebrate cells, its underlying mechanisms remain poorly understood. Here, we report the crystal structure of a human condensin I subcomplex comprising hCAP-G and hCAP-H. hCAP-H binds to the concave surfaces of a harp-shaped HEAT repeat domain of hCAP-G. A physical interaction between hCAP-G and hCAP-H is indeed essential for mitotic chromosome assembly recapitulated in Xenopus egg cell-free extracts. Furthermore, this study reveals that the human CAP-G-H subcomplex has the ability to interact with not only a double-stranded DNA, but also a single-stranded DNA, implicating potential, functional divergence of the vertebrate condensin I complex in mitotic chromosome assembly.

cell biology↗

bioRxiv2018

Differential expression and homotypic enrichment of a classic Cadherin directs tissue-level contractile asymmetry during neural tube closure

Embryos pattern force generation at tissue boundaries during morphogenesis, but how they do so remains poorly understood. Here we show how tissue-specific expression of the type II cadherin, Cadherin2 (hereafter Cad2), patterns actomyosin contractility along the neural/epidermal (Ne/Epi) boundary to drive zippering and neural tube closure in the basal chordate, Ciona robusta. Cad2 is differentially expressed and homotypically enriched in neural cells along the Ne/Epi boundary, where RhoA and Myosin are activated during zipper progression. Equalizing Cad2 expression across the Ne/Epi boundary inhibits RhoA/Myosin activation and zipper progression, while creating ectopic Cad2 expression boundaries is sufficient to direct RhoA/Myosin activity to those boundaries. We show that Cad2 polarizes RhoA activity by sequestering the Rho GTPase activating protein, Gap21/23, to homotypic junctions, which in turn redirects RhoA/Myosin activity to heterotypic Ne/Epi junctions. By activating Myosin II along Ne/Epi junctions ahead of zipper and inhibiting Myosin II at new Ne/Ne junctions behind zipper, Cad2 promotes tissue level contractile asymmetry to drive zipper progression.

developmental biology↗