scholarly journals Investigations of human myosin VI targeting using optogenetically-controlled cargo loading

2016 ◽  
Author(s):  
Alexander R. French ◽  
Tobin R. Sosnick ◽  
Ronald S. Rock
Keyword(s):  
Actin Filaments ◽  
Cancer Metastasis ◽  
Broad Class ◽  
Activation Mechanism ◽  
Myosin Vi ◽  
Cargo Protein ◽  
Multiple Protein ◽  
Pointed End

AbstractMyosins play countless critical roles in the cell, each requiring it to be activated at a specific location and time. To control myosin VI with this specificity, we created a novel optogenetic tool for activating myosin VI by fusing the light-sensitive Avena sativa phototropin1 LOV2 domain to a peptide from Dab2 (LOVDab), a myosin VI cargo protein. Our approach harnesses the native targeting and activation mechanism of myosin VI, allowing direct inferences on myosin VI function. LOVDab robustly recruits human full length myosin VI to various organelles in vivo and hinders peroxisome motion in a light-controllable manner. LOVDab also activates myosin VI in an in vitro gliding filament assay. Our data suggest that protein and lipid cargoes cooperate to activate myosin VI, allowing myosin VI to integrate Ca2+, lipid, and protein cargo signals in the cell to deploy in a site-specific manner.Significance StatementMyosins are a broad class of motor proteins that generate force on actin filaments and fulfill contractile, transport, and anchoring roles. Myosin VI, the only myosin to walk toward the pointed end of actin filaments, is implicated in cancer metastasis and deafness. Intriguingly, myosin VI may play both transport and anchoring roles, depending upon where it is activated in the cell. Here we develop an optogenetic tool for studying myosin VI activation with high spatial and temporal resolution. Our approach photoactivates unmodified myosin VI through its native cargo pathway, enabling investigation of motor function and activation partners with minimal perturbation. This approach allows us to detect how and where myosin VI integrates multiple protein and second messenger signals to activate.

scholarly journals Investigations of human myosin VI targeting using optogenetically controlled cargo loading

2017 ◽  
Vol 114 (9) ◽  
pp. E1607-E1616 ◽  
Author(s):  
Alexander R. French ◽  
Tobin R. Sosnick ◽  
Ronald S. Rock
Keyword(s):  
Avena Sativa ◽  
Activation Mechanism ◽  
Myosin Vi ◽  
Site Specific ◽  
Specific Location ◽  
Cargo Protein ◽  
Specific Manner ◽  
A Site

Myosins play countless critical roles in the cell, each requiring it to be activated at a specific location and time. To control myosin VI with this specificity, we created an optogenetic tool for activating myosin VI by fusing the light-sensitive Avena sativa phototropin1 LOV2 domain to a peptide from Dab2 (LOVDab), a myosin VI cargo protein. Our approach harnesses the native targeting and activation mechanism of myosin VI, allowing direct inferences on myosin VI function. LOVDab robustly recruits human full-length myosin VI to various organelles in vivo and hinders peroxisome motion in a light-controllable manner. LOVDab also activates myosin VI in an in vitro gliding filament assay. Our data suggest that protein and lipid cargoes cooperate to activate myosin VI, allowing myosin VI to integrate Ca2+, lipid, and protein cargo signals in the cell to deploy in a site-specific manner.

Understanding Biological Structures Through Exploring the Mechanical Response of Cell-Like Systems

2008 ◽  
Author(s):  
Ying Zhang ◽  
Philip R. LeDuc
Keyword(s):  
Actin Cytoskeleton ◽  
Actin Filaments ◽  
Living Cell ◽  
Cellular Response ◽  
Cancer Metastasis ◽  
Cell Structure ◽  
Polymer Physics ◽  
Wide Range

The actin cytoskeleton provides mechanical support for the cell and influences activities such as cancer metastasis and chemotaxis. While their mechanical responses have been studied in vivo and in vitro, understanding the link between these two forms remains challenging. To explore this gap and further understand cell structure, we reconstructed the cell cytoskeleton in a membrane-like spherical liposome to mimic the cellular environment; this enables an artificial “cell like” system. Through this approach, we are pursuing a path to compare in vitro mechanics from a polymer physics perspective of individual actin filaments with the in vivo mechanics of a living cell [1]. A living cell contains many organelles, which are in a highly packed environment and require significant organization to function. The actin cytoskeleton provides both structural and organizational regulation that is essential for cellular response. Here, we first encapsulated G-actin into giant unilamellar vesicles through an electroformation technique and then polymerized them into actin filaments (F-actin) within individual vesicles. To probe their conformation, we visualized these vesicles with fluorescence and laser scanning confocal microscopy. We then used a tapping mode atomic force microscopy to determine the mechanical properties of these cell-like systems. These results provide insight into a wide range of fields and studies including polymer physics, cell biology, and biotechnology.

scholarly journals How Listeria exploits host cell actin to form its own cytoskeleton. II. Nucleation, actin filament polarity, filament assembly, and evidence for a pointed end capper.

1992 ◽  
Vol 118 (1) ◽  
pp. 83-93 ◽  
Author(s):  
L G Tilney ◽  
D J DeRosier ◽  
A Weber ◽  
M S Tilney
Keyword(s):  
Host Cell ◽  
Actin Filaments ◽  
Critical Concentration ◽  
Filament Assembly ◽  
Tightly Coupled ◽  
Pointed End ◽  
Phagosomal Membrane ◽  
Simple Picture

After Listeria, a bacterium, is phagocytosed by a macrophage, it dissolves the phagosomal membrane and enters the cytoplasm. The Listeria than nucleates actin filaments from its surface. These newly assembled actin filaments show unidirectional polarity with their barbed ends associated with the surface of the Listeria. Using actin concentrations below the pointed end critical concentration we find that filament elongation must be occurring by monomers adding to the barbed ends, the ends associated with the Listerial surface. If Listeria with tails are incubated in G actin under polymerizing conditions, the Listeria is translocated away from its preformed tail by the elongation of filaments attached to the Listeria. This experiment and others tell us that in vivo filament assembly must be tightly coupled to filament capping and cross-bridging so that if one process outstrips another, chaos ensues. We also show that the actin filaments in the tail are capped on their pointed ends which inhibits further elongation and/or disassembly in vitro. From these results we suggest a simple picture of how Listeria competes effectively for host cell actin. When Listeria secretes a nucleator, the host's actin subunits polymerize into a filament. Host cell machinery terminate the assembly leaving a short filament. Listeria overcomes the host control by nucleating new filaments and thus many short filaments assemble. The newest filaments push existing ones into a growing tail. Thus the competition is between nucleation of filaments caused by Listeria and the filament terminators produced by the host.

scholarly journals Farnesyl dimethyl chromanol targets colon cancer stem cells and prevents colorectal cancer metastasis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kazim Husain ◽  
Domenico Coppola ◽  
Chung S. Yang ◽  
Mokenge P. Malafa
Keyword(s):  
Colorectal Cancer ◽  
Colon Cancer ◽  
Cancer Metastasis ◽  
Annexin V ◽  
Cancer Cell Growth ◽  
Ki 67 ◽  
Sox2 Expression ◽  
Tumour Metastasis

AbstractThe activation and growth of tumour-initiating cells with stem-like properties in distant organs characterize colorectal cancer (CRC) growth and metastasis. Thus, inhibition of colon cancer stem cell (CCSC) growth holds promise for CRC growth and metastasis prevention. We and others have shown that farnesyl dimethyl chromanol (FDMC) inhibits cancer cell growth and induces apoptosis in vitro and in vivo. We provide the first demonstration that FDMC inhibits CCSC viability, survival, self-renewal (spheroid formation), pluripotent transcription factors (Nanog, Oct4, and Sox2) expression, organoids formation, and Wnt/β-catenin signalling, as evidenced by comparisons with vehicle-treated controls. In addition, FDMC inhibits CCSC migration, invasion, inflammation (NF-kB), angiogenesis (vascular endothelial growth factor, VEGF), and metastasis (MMP9), which are critical tumour metastasis processes. Moreover, FDMC induced apoptosis (TUNEL, Annexin V, cleaved caspase 3, and cleaved PARP) in CCSCs and CCSC-derived spheroids and organoids. Finally, in an orthotopic (cecum-injected CCSCs) xenograft metastasis model, we show that FDMC significantly retards CCSC-derived tumour growth (Ki-67); inhibits inflammation (NF-kB), angiogenesis (VEGF and CD31), and β-catenin signalling; and induces apoptosis (cleaved PARP) in tumour tissues and inhibits liver metastasis. In summary, our results demonstrate that FDMC inhibits the CCSC metastatic phenotype and thereby supports investigating its ability to prevent CRC metastases.

scholarly journals Cofactor bypass variants reveal a conformational control mechanism governing cell wall polymerase activity

2016 ◽  
Vol 113 (17) ◽  
pp. 4788-4793 ◽  
Author(s):  
Monica Markovski ◽  
Jessica L. Bohrhunter ◽  
Tania J. Lupoli ◽  
Tsuyoshi Uehara ◽  
Suzanne Walker ◽  
...  
Keyword(s):  
Polymerase Activity ◽  
Activation Mechanism ◽  
Phenotypic Analysis ◽  
Outer Membranes ◽  
Division Site ◽  
Physiological Relevance ◽  
Membrane Lipoproteins ◽  
Shed Light

To fortify their cytoplasmic membrane and protect it from osmotic rupture, most bacteria surround themselves with a peptidoglycan (PG) exoskeleton synthesized by the penicillin-binding proteins (PBPs). As their name implies, these proteins are the targets of penicillin and related antibiotics. We and others have shown that the PG synthases PBP1b and PBP1a ofEscherichia colirequire the outer membrane lipoproteins LpoA and LpoB, respectively, for their in vivo function. Although it has been demonstrated that LpoB activates the PG polymerization activity of PBP1b in vitro, the mechanism of activation and its physiological relevance have remained unclear. We therefore selected for variants of PBP1b (PBP1b*) that bypass the LpoB requirement for in vivo function, reasoning that they would shed light on LpoB function and its activation mechanism. Several of these PBP1b variants were isolated and displayed elevated polymerization activity in vitro, indicating that the activation of glycan polymer growth is indeed one of the relevant functions of LpoB in vivo. Moreover, the location of amino acid substitutions causing the bypass phenotype on the PBP1b structure support a model in which polymerization activation proceeds via the induction of a conformational change in PBP1b initiated by LpoB binding to its UB2H domain, followed by its transmission to the glycosyl transferase active site. Finally, phenotypic analysis of strains carrying a PBP1b* variant revealed that the PBP1b–LpoB complex is most likely not providing an important physical link between the inner and outer membranes at the division site, as has been previously proposed.

scholarly journals AHA1 upregulates IDH1 and metabolic activity to promote growth and metastasis and predicts prognosis in osteosarcoma

2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Diwei Zheng ◽  
Weihai Liu ◽  
Wenlin Xie ◽  
Guanyu Huang ◽  
Qiwei Jiang ◽  
...  
Keyword(s):  
Metabolic Activity ◽  
Cancer Metabolism ◽  
Cancer Metastasis ◽  
Isocitrate Dehydrogenase 1 ◽  
Cell Growth And Migration ◽  
And Migration ◽  
The Relationship ◽  
Common Primary Malignant Bone

AbstractOsteosarcoma (OS) is the most common primary malignant bone tumor in children and adolescents. Although activator of HSP90 ATPase activity 1 (AHA1) is reported to be a potential oncogene, its role in osteosarcoma progression remains largely unclear. Since metabolism reprogramming is involved in tumorigenesis and cancer metastasis, the relationship between AHA1 and cancer metabolism is unknown. In this study, we found that AHA1 is significantly overexpressed in osteosarcoma and related to the prognosis of osteosarcoma patients. AHA1 promotes the growth and metastasis of osteosarcoma both in vitro and in vivo. Mechanistically, AHA1 upregulates the metabolic activity to meet cellular bioenergetic needs in osteosarcoma. Notably, we identified that isocitrate dehydrogenase 1 (IDH1) is a novel client protein of Hsp90-AHA1. Furthermore, the IDH1 protein level was positively correlated with AHA1 in osteosarcoma. And IDH1 overexpression could partially reverse the effect of AHA1 knockdown on cell growth and migration of osteosarcoma. Moreover, high IDH1 level was also associated with poor prognosis of osteosarcoma patients. This study demonstrates that AHA1 positively regulates IDH1 and metabolic activity to promote osteosarcoma growth and metastasis, which provides novel prognostic biomarkers and promising therapeutic targets for osteosarcoma patients.

scholarly journals Epigenetic control via allosteric regulation of mammalian protein arginine methyltransferases

2017 ◽  
Vol 114 (38) ◽  
pp. 10101-10106 ◽  
Author(s):  
Kanishk Jain ◽  
Cyrus Y. Jin ◽  
Steven G. Clarke
Keyword(s):  
Cancer Metastasis ◽  
Gene Activation ◽  
Kinetic Studies ◽  
Arginine Methylation ◽  
Histone H4 ◽  
Protein Arginine Methyltransferases ◽  
Wide Range ◽  
Substrate Concentrations

Arginine methylation on histones is a central player in epigenetics and in gene activation and repression. Protein arginine methyltransferase (PRMT) activity has been implicated in stem cell pluripotency, cancer metastasis, and tumorigenesis. The expression of one of the nine mammalian PRMTs, PRMT5, affects the levels of symmetric dimethylarginine (SDMA) at Arg-3 on histone H4, leading to the repression of genes which are related to disease progression in lymphoma and leukemia. Another PRMT, PRMT7, also affects SDMA levels at the same site despite its unique monomethylating activity and the lack of any evidence for PRMT7-catalyzed histone H4 Arg-3 methylation. We present evidence that PRMT7-mediated monomethylation of histone H4 Arg-17 regulates PRMT5 activity at Arg-3 in the same protein. We analyzed the kinetics of PRMT5 over a wide range of substrate concentrations. Significantly, we discovered that PRMT5 displays positive cooperativity in vitro, suggesting that this enzyme may be allosterically regulated in vivo as well. Most interestingly, monomethylation at Arg-17 in histone H4 not only raised the general activity of PRMT5 with this substrate, but also ameliorated the low activity of PRMT5 at low substrate concentrations. These kinetic studies suggest a biochemical explanation for the interplay between PRMT5- and PRMT7-mediated methylation of the same substrate at different residues and also suggest a general model for regulation of PRMTs. Elucidating the exact relationship between these two enzymes when they methylate two distinct sites of the same substrate may aid in developing therapeutics aimed at reducing PRMT5/7 activity in cancer and other diseases.

scholarly journals lncRNA NORAD Contributes to Colorectal Cancer Progression by Inhibition of miR-202-5p

2018 ◽  
Vol 26 (9) ◽  
pp. 1411-1418 ◽  
Author(s):  
Jie Zhang ◽  
Xiao-Yan Li ◽  
Ping Hu ◽  
Yuan-Sheng Ding
Keyword(s):  
Colorectal Cancer ◽  
Cancer Progression ◽  
Noncoding Rna ◽  
Cancer Metastasis ◽  
Cellular Proliferation ◽  
Inhibitory Effect ◽  
Migration And Invasion ◽  
Competing Endogenous Rna

Previous study indicates that long noncoding RNA NORAD could serve as a competing endogenous RNA to pancreatic cancer metastasis. However, its role in colorectal cancer (CRC) needs to be investigated. In the present study, we found that the expression of NORAD was significantly upregulated in CRC tissues. Furthermore, the expression of NORAD was positively related with CRC metastasis and patients’ poor prognosis. Knockdown of NORAD markedly inhibited CRC cell proliferation, migration, and invasion but induced cell apoptosis in vitro. In vivo experiments also indicated an inhibitory effect of NORAD on tumor growth. Mechanistically, we found that NORAD served as a competing endogenous RNA for miR-202-5p. We found that there was an inverse relationship between the expression of NORAD and miR-202-5p in CRC tissues. Moreover, overexpression of miR-202-5p in SW480 and HCT116 cells significantly inhibited cellular proliferation, migration, and invasion. Taken together, our study demonstrated that the NORAD/miR-202-5p axis plays a pivotal function on CRC progression.

scholarly journals The ER v-SNAREs are required for GPI-anchored protein sorting from other secretory proteins upon exit from the ER

2003 ◽  
Vol 162 (3) ◽  
pp. 403-412 ◽  
Author(s):  
Pierre Morsomme ◽  
Cristina Prescianotto-Baschong ◽  
Howard Riezman
Keyword(s):  
Golgi Complex ◽  
Protein Sorting ◽  
Secretory Proteins ◽  
Rab Gtpase ◽  
Cargo Protein ◽  
Conserved Oligomeric Golgi Complex ◽  
Sorting Process ◽  
Conserved Oligomeric Golgi

Glycosylphosphatidylinositol (GPI)-anchored proteins exit the ER in distinct vesicles from other secretory proteins, and this sorting event requires the Rab GTPase Ypt1p, tethering factors Uso1p, and the conserved oligomeric Golgi complex. Here we show that proper sorting depended on the vSNAREs, Bos1p, Bet1p, and Sec22p. However, the t-SNARE Sed5p was not required for protein sorting upon ER exit. Moreover, the sorting defect observed in vitro with bos1–1 extracts was also observed in vivo and was visualized by EM. Finally, transport and maturation of the GPI-anchored protein Gas1p was specifically affected in a bos1–1 mutant at semirestrictive temperature. Therefore, we propose that v-SNAREs are part of the cargo protein sorting machinery upon exit from the ER and that a correct sorting process is necessary for proper maturation of GPI-anchored proteins.

scholarly journals Knockdown of ARK5 Expression Suppresses Invasion and Metastasis of Gastric Cancer

2017 ◽  
Vol 42 (3) ◽  
pp. 1025-1036 ◽  
Author(s):  
Dehu Chen ◽  
Guiyuan Liu ◽  
Ning Xu ◽  
Xiaolan You ◽  
Haihua Zhou ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Western Blot ◽  
Cancer Metastasis ◽  
Epithelial Mesenchymal Transition ◽  
Migration And Invasion ◽  
Invasion And Metastasis ◽  
Ags Cells ◽  
Mesenchymal Transition

Background/Aims: Gastric cancer (GC) is a common and lethal malignancy, and AMP-activated protein kinase-related kinase 5 (ARK5) has been discovered to promote cancer metastasis in certain types of cancer. In this study, we explored the role of ARK5 in GC invasion and metastasis. Methods: ARK5 and epithelial-mesenchymal transition (EMT)-related markers were determined by immunohistochemistry and western blot in GC specimens. Other methods including stably transfected against ARK5 into SGC7901 and AGS cells, western blot, migration and invasion assays in vitro and nude mice tumorigenicity in vivo were also employed. Results: The results demonstrated that ARK5 expression was increased and positively correlated with metastasis, EMT-related markers and poor prognosis in patients with GC. Knockdown of ARK5 expression remarkably suppressed GC cells invasion and metastasis via regulating EMT, rather than proliferation in vitro and in vivo. And knockdown of ARK5 expression in GC cells resulted in the down-regulation of the mTOR/p70S6k signals, Slug and SIP1. Conclusion: The elevated ARK5 expression was closely associated with cancer metastasis and patient survival, and it seemed to function in GC cells migration and invasion via EMT alteration, together with the alteration of the mTOR/p70S6k signals, Slug and SIP1, thus providing a potential therapeutic target for GC.

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