scholarly journals In-frame deletion of SPECC1L microtubule binding domain results in embryonic tissue movement and fusion defects

2021 ◽  
Author(s):  
Jeremy P. Goering ◽  
Luke W. Wenger ◽  
Marta Stetsiv ◽  
Michael Moedritzer ◽  
Everett G. Hall ◽  
...  
Keyword(s):  
Subcellular Localization ◽  
Cleft Palate ◽  
Coiled Coil ◽  
Cell Cortex ◽  
Cell Alignment ◽  
Tissue Fusion ◽  
Microtubule Binding Domain ◽  
Tissue Movement ◽  
Microtubule Networks ◽  
Muscle Myosin

AbstractEmbryonic morphogenesis of the neural tube, palate, ventral body wall and optic fissure require precise sequence of tissue movement and fusion, which if incomplete, leads to anencephaly/exencephaly, cleft palate, omphalocele and coloboma, respectively. These are genetically heterogeneous birth defects, so there is a continued need to identify etiologic genes. Patients with autosomal dominant SPECC1L mutations show syndromic malformations, including hypertelorism, cleft palate and omphalocele. These SPECC1L mutations cluster in the second coiled-coil domain (CCD2), which facilitates association with microtubules. To study SPECC1L function in mice, we first generated a null allele (Specc1lΔEx4) lacking the entire SPECC1L protein. Homozygous mutants for these truncations died perinatally without cleft palate or exencephaly. Given the clustering of human mutations in CCD2, we hypothesized that targeted perturbation of CCD2 may be required. Indeed, homozygotes for in-frame deletions involving CCD2 (Specc1lΔCCD2) resulted in ~50% exencephaly and ~50% cleft palate. Interestingly, these two phenotypes are never observed in the same embryo. Examination of embryos with and without exencephaly revealed that the oral cavity was narrower in exencephalic embryos, which allowed palatal shelves to elevate despite their defect. In contrast to an evenly distributed subcellular expression pattern, mutant SPECC1L-ΔCCD2 protein showed abnormal subcellular localization, decreased overlap with microtubules, increased actin bundles, and dislocated non-muscle myosin II to the cell cortex. Thus, we show that perturbations of CCD2 in the context of full SPECC1L protein affects tissue fusion dynamics, indicating that human SPECC1L CCD2 mutations are gain-of-function. Improper SPECC1L subcellular localization appears to disrupt connections between actomyosin and microtubule networks, which in turn may affect cell alignment and coordinate movement during tissue morphogenesis.

scholarly journals The dynein cortical anchor Num1 activates dynein motility by relieving Pac1/LIS1-mediated inhibition

2015 ◽  
Vol 211 (2) ◽  
pp. 309-322 ◽  
Author(s):  
Lindsay G. Lammers ◽  
Steven M. Markus
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Budding Yeast ◽  
Coiled Coil ◽  
Binding Domain ◽  
Cell Cortex ◽  
Wild Type ◽  
Microtubule Binding ◽  
Coiled Coil Domain ◽  
Microtubule Binding Domain

Cortically anchored dynein orients the spindle through interactions with astral microtubules. In budding yeast, dynein is offloaded to Num1 receptors from microtubule plus ends. Rather than walking toward minus ends, dynein remains associated with plus ends due in part to its association with Pac1/LIS1, an inhibitor of dynein motility. The mechanism by which dynein is switched from “off” at the plus ends to “on” at the cell cortex remains unknown. Here, we show that overexpression of the coiled-coil domain of Num1 specifically depletes dynein–dynactin–Pac1/LIS1 complexes from microtubule plus ends and reduces dynein-Pac1/LIS1 colocalization. Depletion of dynein from plus ends requires its microtubule-binding domain, suggesting that motility is required. An enhanced Pac1/LIS1 affinity mutant of dynein or overexpression of Pac1/LIS1 rescues dynein plus end depletion. Live-cell imaging reveals minus end–directed dynein–dynactin motility along microtubules upon overexpression of the coiled-coil domain of Num1, an event that is not observed in wild-type cells. Our findings indicate that dynein activity is directly switched “on” by Num1, which induces Pac1/LIS1 removal.

scholarly journals Mcp5, a meiotic cell cortex protein, is required for nuclear movement mediated by dynein and microtubules in fission yeast

2006 ◽  
Vol 173 (1) ◽  
pp. 27-33 ◽  
Author(s):  
Takamune T. Saito ◽  
Daisuke Okuzaki ◽  
Hiroshi Nojima
Keyword(s):  
Fission Yeast ◽  
Meiotic Prophase ◽  
Coiled Coil ◽  
Cell Cortex ◽  
Ph Domain ◽  
Nuclear Movement ◽  
Recombination Rates ◽  
Homologous Chromosome Pairing ◽  
Cortical Localization ◽  
Pulling Force

During meiotic prophase I of the fission yeast Schizosaccharomyces pombe, oscillatory nuclear movement occurs. This promotes homologous chromosome pairing and recombination and involves cortical dynein, which plays a pivotal role by generating a pulling force with the help of an unknown dynein anchor. We show that Mcp5, the homologue of the budding yeast dynein anchor Num1, may be this putative dynein anchor. mcp5+ is predominantly expressed during meiotic prophase, and GFP-Mcp5 localizes at the cell cortex. Moreover, the mcp5Δ strain lacks the oscillatory nuclear movement. Accordingly, homologous pairing and recombination rates of the mcp5Δ strain are significantly reduced. Furthermore, the cortical localization of dynein heavy chain 1 appears to be reduced in mcp5Δ cells. Finally, the full function of Mcp5 requires its coiled-coil and pleckstrin homology (PH) domains. Our results suggest that Mcp5 localizes at the cell cortex through its PH domain and functions as a dynein anchor, thereby facilitating nuclear oscillation.

scholarly journals Cleft lip with or without cleft palate: implication of the heavy chain of non-muscle myosin IIA

2007 ◽  
Vol 44 (6) ◽  
pp. 387-392 ◽  
Author(s):  
M. Martinelli ◽  
M. Di Stazio ◽  
L. Scapoli ◽  
J. Marchesini ◽  
F. Di Bari ◽  
...  
Keyword(s):  
Cleft Palate ◽  
Heavy Chain ◽  
Cleft Lip ◽  
Myosin Iia ◽  
Muscle Myosin

scholarly journals Dynamic accumulation of a helper NLR at the plant-pathogen interface underpins pathogen recognition

2021 ◽  
Author(s):  
Cian Duggan ◽  
Eleonora Moratto ◽  
Zachary Savage ◽  
Eranthika Hamilton ◽  
Hiroaki Adachi ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Immune Responses ◽  
Subcellular Localization ◽  
Plant Pathogen ◽  
Coiled Coil ◽  
Dynamic Changes ◽  
Pathogen Effectors ◽  
Mediate Response ◽  
Potato Famine ◽  
Irish Potato Famine

Plants employ sensor-helper pairs of NLR immune receptors to recognize pathogen effectors and activate immune responses. Yet the subcellular localization of NLRs pre- and post- activation during pathogen infection remains poorly known. Here we show that NRC4, from the 'NRC' solanaceous helper NLR family, undergoes dynamic changes in subcellular localization by shuttling to and from the plant-pathogen haustorium interface established during infection by the Irish potato famine pathogen Phytophthora infestans. Specifically, prior to activation, NRC4 accumulates at the extra-haustorial membrane (EHM), presumably to mediate response to perihaustorial effectors, that are recognized by NRC4-dependent sensor NLRs. However not all NLRs accumulate at the EHM, as the closely related helper NRC2, and the distantly related ZAR1, did not accumulate at the EHM. NRC4 required an intact N- terminal coiled coil domain to accumulate at the EHM, whereas the functionally conserved MADA motif implicated in cell death activation and membrane insertion was dispensable for this process. Strikingly, a constitutively autoactive NRC4 mutant did not accumulate at the EHM and showed punctate distribution that mainly associated with the plasma membrane, suggesting that post-activation, NRC4 probably undergoes a conformation switch to form clusters that do not preferentially associate with the EHM. When NRC4 is activated by a sensor NLR during infection however, NRC4 formed puncta mainly at the EHM and to a lesser extent at the plasma membrane. We conclude that following activation at the EHM, NRC4 may spread to other cellular membranes from its primary site of activation to trigger immune responses.

Inscuteable-dependent apical localization of the microtubule-binding protein Cornetto suggests a role in asymmetric cell division

2001 ◽  
Vol 114 (20) ◽  
pp. 3655-3662 ◽  
Author(s):  
Silvia Bulgheresi ◽  
Elke Kleiner ◽  
Juergen A. Knoblich
Keyword(s):  
Cell Fate ◽  
Mitotic Spindle ◽  
Binding Protein ◽  
Protein Localization ◽  
Apical Cell ◽  
Coiled Coil ◽  
Cell Cortex ◽  
Dependent Manner ◽  
Genetic Redundancy ◽  
Microtubule Binding

Drosophila neuroblasts divide asymmetrically along the apical-basal axis. The Inscuteable protein localizes to the apical cell cortex in neuroblasts from interphase to metaphase, but disappears in anaphase. Inscuteable is required for correct spindle orientation and for asymmetric localization of cell fate determinants to the opposite (basal) cell cortex. Here, we show that Inscuteable also directs asymmetric protein localization to the apical cell cortex during later stages of mitosis. In a two-hybrid screen for Inscuteable-binding proteins, we have identified the coiled-coil protein Cornetto, which shows a highly unusual subcellular distribution in neuroblasts. Although the protein is uniformly distributed in the cytoplasm during metaphase, it concentrates apically in anaphase and forms an apical crescent during telophase in an inscuteable-dependent manner. Upon overexpression, Cornetto localizes to astral microtubules and microtubule spin-down experiments demonstrate that Cornetto is a microtubule-binding protein. After disruption of the actin cytoskeleton, Cornetto localizes with microtubules throughout the cell cycle and decorates the mitotic spindle during metaphase. Our results reveal a novel pattern of asymmetric protein localization in Drosophila neuroblasts and are consistent with a function of Cornetto in anchoring the mitotic spindle during late phases of mitosis, even though our cornetto mutant analysis suggests that this function might be obscured by genetic redundancy.

scholarly journals Num1 anchors mitochondria to the plasma membrane via two domains with different lipid binding specificities

2016 ◽  
Vol 213 (5) ◽  
pp. 513-524 ◽  
Author(s):  
Holly A. Ping ◽  
Lauren M. Kraft ◽  
WeiTing Chen ◽  
Amy E. Nilles ◽  
Laura L. Lackner
Keyword(s):  
Plasma Membrane ◽  
Specific Binding ◽  
Coiled Coil ◽  
Lipid Binding ◽  
General Mechanism ◽  
Cell Cortex ◽  
Pleckstrin Homology Domain ◽  
Binding Domains ◽  
Coiled Coil Domain ◽  
Mitochondrial Distribution

The mitochondria–ER cortex anchor (MECA) is required for proper mitochondrial distribution and functions by tethering mitochondria to the plasma membrane. The core component of MECA is the multidomain protein Num1, which assembles into clusters at the cell cortex. We show Num1 adopts an extended, polarized conformation. Its N-terminal coiled-coil domain (Num1CC) is proximal to mitochondria, and the C-terminal pleckstrin homology domain is associated with the plasma membrane. We find that Num1CC interacts directly with phospholipid membranes and displays a strong preference for the mitochondria-specific phospholipid cardiolipin. This direct membrane interaction is critical for MECA function. Thus, mitochondrial anchoring is mediated by a protein that interacts directly with two different membranes through lipid-specific binding domains, suggesting a general mechanism for interorganelle tethering.

scholarly journals The inv(16) Fusion Protein Associates with Corepressors via a Smooth Muscle Myosin Heavy-Chain Domain

2003 ◽  
Vol 23 (2) ◽  
pp. 607-619 ◽  
Author(s):  
Kristie L. Durst ◽  
Bart Lutterbach ◽  
Tanawan Kummalue ◽  
Alan D. Friedman ◽  
Scott W. Hiebert
Keyword(s):  
Amino Acids ◽  
Smooth Muscle ◽  
Fusion Protein ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Transcriptional Repression ◽  
Coiled Coil ◽  
Smooth Muscle Myosin ◽  
Muscle Myosin ◽  
Repression Domain

ABSTRACT Inversion(16) is one of the most frequent chromosomal translocations found in acute myeloid leukemia (AML), occurring in over 8% of AML cases. This translocation results in a protein product that fuses the first 165 amino acids of core binding factor β to the coiled-coil region of a smooth muscle myosin heavy chain (CBFβ/SMMHC). CBFβ interacts with AML1 to form a heterodimer that binds DNA; this interaction increases the affinity of AML1 for DNA. The CBFβ/SMMHC fusion protein cooperates with AML1 to repress the transcription of AML1-regulated genes. We show that CBFβ/SMMHC contains a repression domain in the C-terminal 163 amino acids of the SMMHC region that is required for inv(16)-mediated transcriptional repression. This minimal repression domain is sufficient for the association of CBFβ/SMMHC with the mSin3A corepressor. In addition, the inv(16) fusion protein specifically associates with histone deacetylase 8 (HDAC8). inv(16)-mediated repression is sensitive to HDAC inhibitors. We propose a model whereby the inv(16) fusion protein associates with AML1 to convert AML1 into a constitutive transcriptional repressor.

The CBFb-SMMHC Oncoprotein Inhibits Binding of the Runx1 Runt Domain to DNA.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2709-2709
Author(s):  
John H. Bushweller ◽  
Stephen M. Lukasik ◽  
Nancy A. Speck
Keyword(s):  
Dna Binding ◽  
Fetal Liver ◽  
Chromosomal Rearrangements ◽  
Coiled Coil ◽  
Embryonic Lethality ◽  
Runt Domain ◽  
Viable Approach ◽  
Muscle Myosin ◽  
Silencer Element

Abstract Core-binding factors (CBFs) are heterodimeric transcriptional factors consisting of a DNA-binding Runx1 (CBFα) subunit and a CBFβ subunit. Cbfβ allosterically increases the affinity of Runx1 for DNA ~2.5 fold. CBF subunits are encoded by four genes in mammals. RUNX1 (AML1), RUNX2, and RUNX3 encode for CBFα subunits, and CBFB encodes the CBFβ subunit. Homozygous disruption of either the Runx1 or the Cbfb genes in mice results in essentially identical phenotypes: midgestation embryonic lethality accompanied by extensive hemorrhaging and a profound block at the fetal liver stage of hematopoiesis. In humans, chromosomal rearrangements that disrupt the Runx1 and CBFB genes are associated with a significant percentage of leukemias. CBFβ is disrupted in acute myeloid leukemia by inv(16)(p13;q22), t(16;16), and del(16)(q22). These translocations result in the production of novel fusion proteins containing most of the CBFβ protein fused to the C-terminal coiled-coil domain from smooth muscle myosin heavy chain (SMMHC) encoded by the MYH11 gene. A knock-in of the CBFB-MYH11 allele in mice resulted in embryonic lethality with a profound block in hematopoietic development, the same phenotype observed for the Runx1 and Cbfb knockouts. We recently demonstrated that the CBFβ-SMMHC fusion protein binds to the DNA binding Runt domain from Runx1 with both higher affinity and altered stoichiometry relative to native CBFβ. We also provided NMR-based evidence for multiple sites of contact between Runx1 and CBFβ-SMMHC, proving the role of the SMMHC sequence in creating this altered affinity. Here we demonstrate that CBFβ-SMMHC inhibits DNA binding of the Runx1 Runt domain by ~6-fold for the CD4 dual-site silencer element. Cross-saturation NMR mapping on the Runt domain in complex with CBFβ-SMMHC reveals that the SMMHC portion of the oncoprotein makes contacts with β-strands 1 and 2 in the Runt domain. We propose that the inhibition of DNA-binding and increased affinity combine to mediate the dysregulation of Runx-regulated genes caused by CBFβ-SMMHC. These results also clearly suggest that targeting of the CBFβ-SMMHC protein for drug development may well be a viable approach for the treatment of the associated leukemia.

scholarly journals Mcp4, a Meiotic Coiled-Coil Protein, Plays a Role in F-Actin Positioning during Schizosaccharomyces pombe Meiosis

2007 ◽  
Vol 6 (6) ◽  
pp. 971-983 ◽  
Author(s):  
Ayami Ohtaka ◽  
Daisuke Okuzaki ◽  
Takamune T. Saito ◽  
Hiroshi Nojima
Keyword(s):  
Subcellular Localization ◽  
Schizosaccharomyces Pombe ◽  
Coiled Coil ◽  
Microscopic Analysis ◽  
Specific Protein ◽  
Membrane Formation ◽  
Meiotic Progression ◽  
Specific Proteins ◽  
Fluorescence Microscopic Analysis

ABSTRACT Some meiosis-specific proteins of Schizosaccharomyces pombe harbor coiled-coil motifs and play essential roles in meiotic progression. Here we describe Mcp4, a novel meiosis-specific protein whose expression is abruptly induced at the horsetail phase and which remains expressed until sporulation is finished. Fluorescence microscopic analysis revealed that Mcp4 alters its subcellular localization during meiosis in a manner that partially resembles the movement of F-actin during meiosis. Mcp4 and F-actin never colocalize; rather, they are located in a side-by-side manner. When forespore membrane formation begins at metaphase II, the Mcp4 signals assemble at the lagging face of the dividing nuclei. At this stage, they are sandwiched between F-actin and the nucleus. Mcp4, in turn, appears to sandwich F-actin with Meu14. In mcp4Δ cells at anaphase II, the F-actin, which is normally dumbbell-shaped, adopts an abnormal balloon shape. Spores of mcp4Δ cells were sensitive to NaCl, although their shape and viability were normal. Taken together, we conclude that Mcp4 plays a role in the accurate positioning of F-actin during S. pombe meiosis.

scholarly journals Hydrophobicity variations along the surface of the coiled-coil rod may mediate striated muscle myosin assembly in Caenorhabditis elegans.

1996 ◽  
Vol 135 (2) ◽  
pp. 371-382 ◽  
Author(s):  
P E Hoppe ◽  
R H Waterston
Keyword(s):  
Caenorhabditis Elegans ◽  
Striated Muscle ◽  
Coiled Coil ◽  
Thick Filament ◽  
Surface Hydrophobicity ◽  
Filament Assembly ◽  
Thick Filaments ◽  
Characteristic Variation ◽  
Muscle Myosin

Caenorhabditis elegans body wall muscle contains two isoforms of myosin heavy chain, MHC A and MHC B, that differ in their ability to initiate thick filament assembly. Whereas mutant animals that lack the major isoform, MHC B, have fewer thick filaments, mutant animals that lack the minor isoform, MHC A, contain no normal thick filaments. MHC A, but not MHC B, is present at the center of the bipolar thick filament where initiation of assembly is thought to occur (Miller, D.M.,I. Ortiz, G.C. Berliner, and H.F. Epstein. 1983. Cell. 34:477-490). We mapped the sequences that confer A-specific function by constructing chimeric myosins and testing them in vivo. We have identified two distinct regions of the MHC A rod that are sufficient in chimeric myosins for filament initiation function. Within these regions, MHC A displays a more hydrophobic rod surface, making it more similar to paramyosin, which forms the thick filament core. We propose that these regions play an important role in filament initiation, perhaps mediating close contacts between MHC A and paramyosin in an antiparallel arrangement at the filament center. Furthermore, our analysis revealed that all striated muscle myosins show a characteristic variation in surface hydrophobicity along the length of the rod that may play an important role in driving assembly and determining the stagger at which dimers associate.

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