scholarly journals Abl and Canoe/Afadin mediate mechanotransduction at tricellular junctions

Science ◽  
2020 ◽  
Vol 370 (6520) ◽  
pp. eaba5528 ◽  
Author(s):  
Huapeng H. Yu ◽  
Jennifer A. Zallen
Keyword(s):  
Cell Adhesion ◽  
Actin Binding ◽  
Mechanical Forces ◽  
Abl Tyrosine Kinase ◽  
Actomyosin Contractility ◽  
Actin Binding Domain ◽  
Epithelial Structure ◽  
Epithelial Remodeling ◽  
Sense And Respond

Epithelial structure is generated by the dynamic reorganization of cells in response to mechanical forces. Adherens junctions transmit forces between cells, but how cells sense and respond to these forces in vivo is not well understood. We identify a mechanotransduction pathway involving the Abl tyrosine kinase and Canoe/Afadin that stabilizes cell adhesion under tension at tricellular junctions in the Drosophila embryo. Canoe is recruited to tricellular junctions in response to actomyosin contractility, and this mechanosensitivity requires Abl-dependent phosphorylation of a conserved tyrosine in the Canoe actin-binding domain. Preventing Canoe tyrosine phosphorylation destabilizes tricellular adhesion, and anchoring Canoe at tricellular junctions independently of mechanical inputs aberrantly stabilizes adhesion, arresting cell rearrangement. These results identify a force-responsive mechanism that stabilizes tricellular adhesion under tension during epithelial remodeling.

Reduced oncogenicity of p190 Bcr/Abl F-actin–binding domain mutants

Blood ◽  
2000 ◽  
Vol 96 (6) ◽  
pp. 2226-2232 ◽  
Author(s):  
Nora Heisterkamp ◽  
Jan Willem Voncken ◽  
Dinithi Senadheera ◽  
Ignacio Gonzalez-Gomez ◽  
Anja Reichert ◽  
...  
Keyword(s):  
Binding Domain ◽  
Actin Binding ◽  
Abl Tyrosine Kinase ◽  
Stage Disease ◽  
Absolute Requirement ◽  
Actin Binding Domain ◽  
High Level ◽  
End Stage ◽  
Cas Proteins

The deregulated Bcr/Abl tyrosine kinase is responsible for the development of Philadelphia (Ph)-positive leukemia in humans. To investigate the significance of the C-terminal Abl actin-binding domain within Bcr/Abl p190 in the development of leukemia/lymphoma in vivo, mutant p190 DNA constructs were used to generate transgenic mice. Eight founder and progeny mice of 5 different lines were monitored for leukemogenesis. Latency was markedly increased and occurrence decreased in the p190 del C lines as compared with nonmutated p190BCR/ABL transgenics. Western blot analysis of involved hematologic tissues of the p190 del C transgenics with end-stage disease showed high-level expression of the transgene and tyrosine phosphorylation of Cbl and Hef1/Cas, proteins previously shown to be affected by Bcr/Abl. These results show that the actin-binding domain of Abl enhances leukemia development but does not appear to be an absolute requirement for leukemogenesis.

Reduced oncogenicity of p190 Bcr/Abl F-actin–binding domain mutants

Blood ◽  
2000 ◽  
Vol 96 (6) ◽  
pp. 2226-2232 ◽  
Author(s):  
Nora Heisterkamp ◽  
Jan Willem Voncken ◽  
Dinithi Senadheera ◽  
Ignacio Gonzalez-Gomez ◽  
Anja Reichert ◽  
...  
Keyword(s):  
Binding Domain ◽  
Actin Binding ◽  
Abl Tyrosine Kinase ◽  
Stage Disease ◽  
Absolute Requirement ◽  
Actin Binding Domain ◽  
High Level ◽  
End Stage ◽  
Cas Proteins

Abstract The deregulated Bcr/Abl tyrosine kinase is responsible for the development of Philadelphia (Ph)-positive leukemia in humans. To investigate the significance of the C-terminal Abl actin-binding domain within Bcr/Abl p190 in the development of leukemia/lymphoma in vivo, mutant p190 DNA constructs were used to generate transgenic mice. Eight founder and progeny mice of 5 different lines were monitored for leukemogenesis. Latency was markedly increased and occurrence decreased in the p190 del C lines as compared with nonmutated p190BCR/ABL transgenics. Western blot analysis of involved hematologic tissues of the p190 del C transgenics with end-stage disease showed high-level expression of the transgene and tyrosine phosphorylation of Cbl and Hef1/Cas, proteins previously shown to be affected by Bcr/Abl. These results show that the actin-binding domain of Abl enhances leukemia development but does not appear to be an absolute requirement for leukemogenesis.

scholarly journals Microtubule Actin Cross-Linking Factor (Macf)

1999 ◽  
Vol 147 (6) ◽  
pp. 1275-1286 ◽  
Author(s):  
Conrad L. Leung ◽  
Dongming Sun ◽  
Min Zheng ◽  
David R. Knowles ◽  
Ronald K.H. Liem
Keyword(s):  
Calcium Binding ◽  
Coiled Coil ◽  
Specific Protein ◽  
Binding Domain ◽  
Full Length ◽  
Actin Binding ◽  
Cross Linking ◽  
Actin Binding Domain

We cloned and characterized a full-length cDNA of mouse actin cross-linking family 7 (mACF7) by sequential rapid amplification of cDNA ends–PCR. The completed mACF7 cDNA is 17 kb and codes for a 608-kD protein. The closest relative of mACF7 is the Drosophila protein Kakapo, which shares similar architecture with mACF7. mACF7 contains a putative actin-binding domain and a plakin-like domain that are highly homologous to dystonin (BPAG1-n) at its NH2 terminus. However, unlike dystonin, mACF7 does not contain a coiled–coil rod domain; instead, the rod domain of mACF7 is made up of 23 dystrophin-like spectrin repeats. At its COOH terminus, mACF7 contains two putative EF-hand calcium-binding motifs and a segment homologous to the growth arrest–specific protein, Gas2. In this paper, we demonstrate that the NH2-terminal actin-binding domain of mACF7 is functional both in vivo and in vitro. More importantly, we found that the COOH-terminal domain of mACF7 interacts with and stabilizes microtubules. In transfected cells full-length mACF7 can associate not only with actin but also with microtubules. Hence, we suggest a modified name: MACF (microtubule actin cross-linking factor). The properties of MACF are consistent with the observation that mutations in kakapo cause disorganization of microtubules in epidermal muscle attachment cells and some sensory neurons.

NUANCE, a giant protein connecting the nucleus and actin cytoskeleton

2002 ◽  
Vol 115 (15) ◽  
pp. 3207-3222 ◽  
Author(s):  
Yen-Yi Zhen ◽  
Thorsten Libotte ◽  
Martina Munck ◽  
Angelika A. Noegel ◽  
Elena Korenbaum
Keyword(s):  
Actin Cytoskeleton ◽  
Transmembrane Domain ◽  
Coiled Coil ◽  
Peripheral Blood Lymphocytes ◽  
Binding Domain ◽  
Actin Binding ◽  
Actin Binding Domain ◽  
Microfilament System

NUANCE (NUcleus and ActiN Connecting Element) was identified as a novel protein with an α-actinin-like actin-binding domain. A human 21.8 kb cDNA of NUANCE spreads over 373 kb on chromosome 14q22.1-q22.3. The cDNA sequence predicts a 796 kDa protein with an N-terminal actin-binding domain, a central coiled-coil rod domain and a predicted C-terminal transmembrane domain. High levels of NUANCE mRNA were detected in the kidney, liver,stomach, placenta, spleen, lymphatic nodes and peripheral blood lymphocytes. At the subcellular level NUANCE is present predominantly at the outer nuclear membrane and in the nucleoplasm. Domain analysis shows that the actin-binding domain binds to Factin in vitro and colocalizes with the actin cytoskeleton in vivo as a GFP-fusion protein. The C-terminal transmembrane domain is responsible for the targeting the nuclear envelope. Thus, NUANCE is the firstα-actinin-related protein that has the potential to link the microfilament system with the nucleus.

scholarly journals Modeling and Experimental Validation of the Binary Complex of the Plectin Actin-binding Domain and the First Pair of Fibronectin Type III (FNIII) Domains of the β4 Integrin

2005 ◽  
Vol 280 (23) ◽  
pp. 22270-22277 ◽  
Author(s):  
Sandy H. M. Litjens ◽  
Kevin Wilhelmsen ◽  
José M. de Pereda ◽  
Anastassis Perrakis ◽  
Arnoud Sonnenberg
Keyword(s):  
Mutational Analysis ◽  
Protein Docking ◽  
Binding Domain ◽  
Actin Binding ◽  
Binary Complex ◽  
Type Iii ◽  
Actin Binding Domain ◽  
Fibronectin Type Iii ◽  
Fibronectin Type

The binding of plectin to the β4 subunit of the α6β4 integrin is a critical step in the formation of hemidesmosomes. An important interaction between these two proteins occurs between the actin-binding domain (ABD) of plectin and the first pair of fibronectin type III (FNIII) domains and a small part of the connecting segment of β4. Previously, a few amino acids, critical for this interaction, were identified in both plectin and β4 and mapped on the crystal structures of the ABD of plectin and the first pair of FNIII domains of β4. In the present study, we used this biochemical information and protein-protein docking calculations to construct a model of the binary complex between these two protein domains. The top scoring computational model predicts that the calponin-homology 1 (CH1) domain of the ABD associates with the first and the second FNIII domains of β4. Our mutational analysis of the residues at the proposed interface of both the FNIII and the CH1 domains is in agreement with the suggested interaction model. Computational simulations to predict protein motions suggest that the exact model of FNIII and plectin CH1 interaction might well differ in detail from the suggested model due to the conformational plasticity of the FNIII domains, which might lead to a closely related but different mode of interaction with the plectin-ABD. Furthermore, we show that Ser-1325 in the connecting segment of β4 appears to be essential for the recruitment of plectin into hemidesmosomes in vivo. This is consistent with the proposed model and previously published mutational data. In conclusion, our data support a model in which the CH1 domain of the plectin-ABD associates with the groove between the two FNIII domains of β4.

scholarly journals Functional recovery of troponin I in a Drosophila heldup mutant after a second site mutation.

1995 ◽  
Vol 6 (11) ◽  
pp. 1433-1441 ◽  
Author(s):  
A Prado ◽  
I Canal ◽  
J A Barbas ◽  
J Molloy ◽  
A Ferrús
Keyword(s):  
Functional Recovery ◽  
Troponin I ◽  
Flight Muscle ◽  
Actin Binding ◽  
Stoichiometric Ratio ◽  
Site Mutation ◽  
Single Nucleotide ◽  
Single Nucleotide Change ◽  
Actin Binding Domain

To identify proteins that interact in vivo with muscle components we have used a genetic approach based on the isolation of suppressors of mutant alleles of known muscle components. We have applied this system to the case of troponin I (TnI) in Drosophila and its mutant allele heldup2 (hdp2). This mutation causes an alanine to valine substitution at position 116 after a single nucleotide change in a constitutive exon. Among the isolated suppressors, one of them results from a second site mutation at the TnI gene itself. Muscles endowed with TnI mutated at both sites support nearly normal myofibrillar structure, perform notably well in wing beating and flight tests, and isolated muscle fibers produce active force. We show that the structural and functional recovery in this suppressor does not result from a change in the stoichiometric ratio of TnI isoforms. The second site suppression is due to a leucine to phenylalanine change within a heptameric leucine string motif adjacent to the actin binding domain of TnI. These data evidence a structural and functional role for the heptameric leucine string that is most noticeable, if not specific, in the indirect flight muscle.

scholarly journals Coronin 1A depletion restores the nuclear stability and viability of Aip1/Wdr1-deficient neutrophils

2019 ◽  
Vol 218 (10) ◽  
pp. 3258-3271
Author(s):  
Charnese Bowes ◽  
Michael Redd ◽  
Malika Yousfi ◽  
Muriel Tauzin ◽  
Emi Murayama ◽  
...  
Keyword(s):  
Cell Death ◽  
Actin Filaments ◽  
Essential Role ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Zebrafish Embryos ◽  
Actomyosin Contractility ◽  
Nuclear Stability

Actin dynamics is central for cells, and especially for the fast-moving leukocytes. The severing of actin filaments is mainly achieved by cofilin, assisted by Aip1/Wdr1 and coronins. We found that in Wdr1-deficient zebrafish embryos, neutrophils display F-actin cytoplasmic aggregates and a complete spatial uncoupling of phospho-myosin from F-actin. They then undergo an unprecedented gradual disorganization of their nucleus followed by eruptive cell death. Their cofilin is mostly unphosphorylated and associated with F-actin, thus likely outcompeting myosin for F-actin binding. Myosin inhibition reproduces in WT embryos the nuclear instability and eruptive death of neutrophils seen in Wdr1-deficient embryos. Strikingly, depletion of the main coronin of leukocytes, coronin 1A, fully restores the cortical location of F-actin, nuclear integrity, viability, and mobility of Wdr1-deficient neutrophils in vivo. Our study points to an essential role of actomyosin contractility in maintaining the integrity of the nucleus of neutrophils and a new twist in the interplay of cofilin, Wdr1, and coronin in regulating F-actin dynamics.

Single-molecule imaging of IQGAP1 regulating actin filament dynamics

2021 ◽  
Author(s):  
Gregory J. Hoeprich ◽  
Amy N. Sinclair ◽  
Shashank Shekhar ◽  
Bruce L. Goode
Keyword(s):  
Cell Adhesion ◽  
Actin Filament ◽  
Single Molecule ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Functional Effect ◽  
Single Filament ◽  
Filament Dynamics ◽  
Do So

IQGAP is a conserved family of actin-binding proteins with essential roles in cell motility, cytokinesis, and cell adhesion, yet there remains a limited understanding of how IQGAP proteins directly influence actin filament dynamics. To close this gap, we used single-molecule and single-filament TIRF microscopy to observe IQGAP regulating actin dynamics in real time. To our knowledge, this is the first study to do so. Our results demonstrate that full-length human IQGAP1 forms dimers that stably bind to actin filament sides and transiently cap barbed ends. These interactions organize filaments into thin bundles, suppress barbed end growth, and inhibit filament disassembly. Surprisingly, each activity depends on distinct combinations of IQGAP1 domains and/or dimerization, suggesting that different mechanisms underlie each functional effect on actin. These observations have important implications for how IQGAP functions as an actin regulator in vivo, and how it may be regulated in different biological settings. [Media: see text] [Media: see text] [Media: see text]

scholarly journals A High-affinity Interaction with ADP-Actin Monomers Underlies the Mechanism and In Vivo Function of Srv2/cyclase-associated Protein

2004 ◽  
Vol 15 (11) ◽  
pp. 5158-5171 ◽  
Author(s):  
Pieta K. Mattila ◽  
Omar Quintero-Monzon ◽  
Jamie Kugler ◽  
James B. Moseley ◽  
Steven C. Almo ◽  
...  
Keyword(s):  
Site Directed Mutagenesis ◽  
Actin Binding ◽  
Actin Monomer ◽  
Nucleotide Exchange ◽  
Actin Organization ◽  
C Terminus ◽  
Actin Binding Domain ◽  
Affinity Interaction

Cyclase-associated protein (CAP), also called Srv2 in Saccharomyces cerevisiae, is a conserved actin monomer-binding protein that promotes cofilin-dependent actin turnover in vitro and in vivo. However, little is known about the mechanism underlying this function. Here, we show that S. cerevisiae CAP binds with strong preference to ADP-G-actin (Kd 0.02 μM) compared with ATP-G-actin (Kd 1.9 μM) and competes directly with cofilin for binding ADP-G-actin. Further, CAP blocks actin monomer addition specifically to barbed ends of filaments, in contrast to profilin, which blocks monomer addition to pointed ends of filaments. The actin-binding domain of CAP is more extensive than previously suggested and includes a recently solved β-sheet structure in the C-terminus of CAP and adjacent sequences. Using site-directed mutagenesis, we define evolutionarily conserved residues that mediate binding to ADP-G-actin and demonstrate that these activities are required for CAP function in vivo in directing actin organization and polarized cell growth. Together, our data suggest that in vivo CAP competes with cofilin for binding ADP-actin monomers, allows rapid nucleotide exchange to occur on actin, and then because of its 100-fold weaker binding affinity for ATP-actin compared with ADP-actin, allows other cellular factors such as profilin to take the handoff of ATP-actin and facilitate barbed end assembly.

Localization of BCR-ABL to F-actin regulates cell adhesion but does not attenuate CML development

Blood ◽  
2003 ◽  
Vol 102 (6) ◽  
pp. 2220-2228 ◽  
Author(s):  
Jason A. Wertheim ◽  
Samanthi A. Perera ◽  
Daniel A. Hammer ◽  
Ruibao Ren ◽  
David Boettiger ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Function Analysis ◽  
Coiled Coil ◽  
Myelogenous Leukemia ◽  
Actin Binding ◽  
Murine Bone Marrow ◽  
Actin Binding Domain ◽  
Transplantation Assay ◽  
Α5β1 Integrin ◽  
Actin Localization

Abstract We have previously found that P210BCR-ABL increases the adhesion of hematopoietic cell lines to fibronectin by a mechanism that is independent of tyrosine kinase activity. To investigate the pathway(s) by which P210BCR-ABL influences cell adhesion, we used a quantitative cell adhesion device that can discern small changes in cell adhesion to assay P210BCR-ABL with mutations in several critical domains. We expressed P210BCR-ABL mutants in 32D myeloblast cells and found that binding to fibronectin is mediated primarily by the α5β1 integrin. We performed a structure/function analysis to map domains important for cell adhesion. Increased adhesion was mediated by 3 domains: (1) the N-terminal coiled-coil domain that facilitates oligomerization and F-actin localization; (2) bcr sequences between aa 163 to 210; and (3) F-actin localization through the C-terminal actin-binding domain of c-abl. We compared our adhesion results with the ability of these mutants to cause a chronic myelogenous leukemia (CML)–like disease in a murine bone marrow transplantation assay and found that adhesion to fibronectin did not correlate with the ability of these mutants to cause CML. Together, our results suggest that F-actin localization may play a pivotal role in modulating adhesion but that it is dispensable for the development of CML.

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