Survey of cancer cell anatomy in non-adhesive confinement reveals a role for filamin-A and fascin-1 in leader bleb-based migration

Cancer cells migrating in confined microenvironments exhibit plasticity of migration modes. Confinement of contractile cells in a non-adhesive environment drives ‘leader bleb-based migration’ (LBBM), morphologically characterized by a long bleb that points in the direction of movement separated from a cell body by a contractile neck. Although cells undergoing LBBM have been visualized within tumors, the organization of organelles and actin regulatory proteins mediating LBBM is unknown. We analyzed the localization of fluorescent organelle-specific markers and actin-associated proteins in human melanoma and osteosarcoma cells undergoing LBBM. We found that organelles from the endo-lysosomal, secretory and metabolic systems as well as the vimentin and microtubule cytoskeletons localized primarily in the cell body, with some endoplasmic reticulum, microtubules, and mitochondria extending into the leader bleb. Overexpression of fluorescent-tagged actin regulatory proteins showed that actin assembly factors localized towards the leader bleb tip, contractility regulators and crosslinkers in the cell body cortex and neck, and crosslinkers additionally throughout the leader bleb. Quantitative analysis showed that excess filamin-A and fascin-1 increased migration speed and persistence, while their depletion by siRNA indicate a requirement in promoting cortical tension and pressure to drive LBBM, indicating the critical role of specific actin crosslinkers in LBBM. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text]

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A proteomic survey of microtubule-associated proteins in a R402H TUBA1A mutant mouse

PLoS Genetics ◽

10.1371/journal.pgen.1009104 ◽

2020 ◽

Vol 16 (11) ◽

pp. e1009104

Author(s):

Ines Leca ◽

Alexander William Phillips ◽

Iris Hofer ◽

Lukas Landler ◽

Lyubov Ushakova ◽

...

Keyword(s):

Neuronal Migration ◽

Molecular Mechanisms ◽

Mutant Mouse ◽

Critical Role ◽

Recurrent Mutation ◽

Intrinsic Defect ◽

Quantitative Mass Spectrometry ◽

Microtubule Associated Proteins ◽

Associated Proteins ◽

A Cell

Microtubules play a critical role in multiple aspects of neurodevelopment, including the generation, migration and differentiation of neurons. A recurrent mutation (R402H) in the α-tubulin gene TUBA1A is known to cause lissencephaly with cerebellar and striatal phenotypes. Previous work has shown that this mutation does not perturb the chaperone-mediated folding of tubulin heterodimers, which are able to assemble and incorporate into the microtubule lattice. To explore the molecular mechanisms that cause the disease state we generated a new conditional mouse line that recapitulates the R402H variant. We show that heterozygous mutants present with laminar phenotypes in the cortex and hippocampus, as well as a reduction in striatal size and cerebellar abnormalities. We demonstrate that homozygous expression of the R402H allele causes neuronal death and exacerbates a cell intrinsic defect in cortical neuronal migration. Microtubule sedimentation assays coupled with quantitative mass spectrometry demonstrated that the binding and/or levels of multiple microtubule associated proteins (MAPs) are perturbed by the R402H mutation including VAPB, REEP1, EZRIN, PRNP and DYNC1l1/2. Consistent with these data we show that the R402H mutation impairs dynein-mediated transport which is associated with a decoupling of the nucleus to the microtubule organising center. Our data support a model whereby the R402H variant is able to fold and incorporate into microtubules, but acts as a gain of function by perturbing the binding of MAPs.

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Differential regulation of mast cell degranulation versus cytokine secretion by the actin regulatory proteins Coronin1a and Coronin1b

Journal of Experimental Medicine ◽

10.1084/jem.20101757 ◽

2011 ◽

Vol 208 (9) ◽

pp. 1777-1787 ◽

Cited By ~ 39

Author(s):

Niko Föger ◽

André Jenckel ◽

Zane Orinska ◽

Kyeong-Hee Lee ◽

Andrew C. Chan ◽

...

Keyword(s):

Mast Cell ◽

Actin Cytoskeleton ◽

Critical Role ◽

Cytokine Secretion ◽

Regulatory Proteins ◽

Actin Binding ◽

Filamentous Actin ◽

Regulatory Pathways ◽

Proinflammatory Mediators ◽

Actin Regulatory Proteins

Mast cell (MC) activation via aggregation of the high affinity IgE receptor (FcεRI) causes degranulation and release of proinflammatory mediators in a process that involves the reorganization of the actin cytoskeleton. However, the regulatory pathways and the molecular links between cytoskeletal changes and MC function are incompletely understood. In this study, we provide genetic evidence for a critical role of the actin-regulatory proteins Coronin1a (Coro1a) and Coro1b on exocytic pathways in MCs: Coro1a−/− bone marrow–derived MCs exhibit increased FcεRI-mediated degranulation of secretory lysosomes but significantly reduced secretion of cytokines. Hyperdegranulation of Coro1a−/− MCs is further augmented by the additional loss of Coro1b. In vivo, Coro1a−/−Coro1b−/− mice displayed enhanced passive cutaneous anaphylaxis. Functional reconstitution assays revealed that the inhibitory effect of Coro1a on MC degranulation strictly correlates with cortical localization of Coro1a, requires its filamentous actin–binding activity, and is regulated by phosphorylation of Ser2 of Coro1a. Thus, coronin proteins, and in turn the actin cytoskeleton, exhibit a functional dichotomy as differential regulators of degranulation versus cytokine secretion in MC biology.

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Self-Assembling Polypeptide Hydrogels as a Platform to Recapitulate the Tumor Microenvironment

Cancers ◽

10.3390/cancers13133286 ◽

2021 ◽

Vol 13 (13) ◽

pp. 3286

Author(s):

Dariusz Lachowski ◽

Carlos Matellan ◽

Ernesto Cortes ◽

Alberto Saiani ◽

Aline F. Miller ◽

...

Keyword(s):

Cell Culture ◽

Tumor Microenvironment ◽

Cancer Cell ◽

Critical Role ◽

Hypoxia Inducible Factor ◽

Pancreatic Cancer Cell Line ◽

Cancer Cell Line ◽

Culture Systems ◽

A Cell

The tumor microenvironment plays a critical role in modulating cancer cell migration, metabolism, and malignancy, thus, highlighting the need to develop in vitro culture systems that can recapitulate its abnormal properties. While a variety of stiffness-tunable biomaterials, reviewed here, have been developed to mimic the rigidity of the tumor extracellular matrix, culture systems that can recapitulate the broader extracellular context of the tumor microenvironment (including pH and temperature) remain comparably unexplored, partially due to the difficulty in independently tuning these parameters. Here, we investigate a self-assembled polypeptide network hydrogel as a cell culture platform and demonstrate that the culture parameters, including the substrate stiffness, extracellular pH and temperature, can be independently controlled. We then use this biomaterial as a cell culture substrate to assess the effect of stiffness, pH and temperature on Suit2 cells, a pancreatic cancer cell line, and demonstrate that these microenvironmental factors can regulate two critical transcription factors in cancer: yes-associated protein 1 (YAP) and hypoxia inducible factor (HIF-1A).

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Annexin A2 in Fibrinolysis, Inflammation and Fibrosis

International Journal of Molecular Sciences ◽

10.3390/ijms22136836 ◽

2021 ◽

Vol 22 (13) ◽

pp. 6836

Author(s):

Hana I. Lim ◽

Katherine A. Hajjar

Keyword(s):

Cell Surface ◽

Tissue Injury ◽

Critical Role ◽

Annexin A2 ◽

Hemorrhagic Disease ◽

Membrane Repair ◽

Endothelial Cell Surface ◽

Human Disorders ◽

A Cell ◽

Organ Fibrosis

As a cell surface tissue plasminogen activator (tPA)-plasminogen receptor, the annexin A2 (A2) complex facilitates plasmin generation on the endothelial cell surface, and is an established regulator of hemostasis. Whereas A2 is overexpressed in hemorrhagic disease such as acute promyelocytic leukemia, its underexpression or impairment may result in thrombosis, as in antiphospholipid syndrome, venous thromboembolism, or atherosclerosis. Within immune response cells, A2 orchestrates membrane repair, vesicle fusion, and cytoskeletal organization, thus playing a critical role in inflammatory response and tissue injury. Dysregulation of A2 is evident in multiple human disorders, and may contribute to the pathogenesis of various inflammatory disorders. The fibrinolytic system, moreover, is central to wound healing through its ability to remodel the provisional matrix and promote angiogenesis. A2 dysfunction may also promote tissue fibrogenesis and end-organ fibrosis.

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The Annexin A2/S100A10 System in Health and Disease: Emerging Paradigms

Journal of Biomedicine and Biotechnology ◽

10.1155/2012/406273 ◽

2012 ◽

Vol 2012 ◽

pp. 1-13 ◽

Cited By ~ 45

Author(s):

Nadia Hedhli ◽

Domenick J. Falcone ◽

Bihui Huang ◽

Gabriela Cesarman-Maus ◽

Rosemary Kraemer ◽

...

Keyword(s):

Serotonin Receptor ◽

Human Cancer ◽

High Titer ◽

Critical Role ◽

Annexin A2 ◽

Avascular Osteonecrosis ◽

Kinase Substrate ◽

New Findings ◽

Human Disorders ◽

Associated Proteins

Since its discovery as a src kinase substrate more than three decades ago, appreciation for the physiologic functions of annexin A2 and its associated proteins has increased dramatically. With its binding partner S100A10 (p11), A2 forms a cell surface complex that regulates generation of the primary fibrinolytic protease, plasmin, and is dynamically regulated in settings of hemostasis and thrombosis. In addition, the complex is transcriptionally upregulated in hypoxia and promotes pathologic neoangiogenesis in the tissues such as the retina. Dysregulation of both A2 and p11 has been reported in examples of rodent and human cancer. Intracellularly, A2 plays a critical role in endosomal repair in postarthroplastic osteolysis, and intracellular p11 regulates serotonin receptor activity in psychiatric mood disorders. In human studies, the A2 system contributes to the coagulopathy of acute promyelocytic leukemia, and is a target of high-titer autoantibodies in patients with antiphospholipid syndrome, cerebral thrombosis, and possibly preeclampsia. Polymorphisms in the humanANXA2gene have been associated with stroke and avascular osteonecrosis of bone, two severe complications of sickle cell disease. Together, these new findings suggest that manipulation of the annexin A2/S100A10 system may offer promising new avenues for treatment of a spectrum of human disorders.

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Cell cycle regulatory proteins in renal disease: role in hypertrophy, proliferation, and apoptosis

AJP Renal Physiology ◽

10.1152/ajprenal.2000.278.4.f515 ◽

2000 ◽

Vol 278 (4) ◽

pp. F515-F529 ◽

Cited By ~ 103

Author(s):

Stuart J. Shankland ◽

Gunter Wolf

Keyword(s):

Cell Cycle ◽

Renal Function ◽

Renal Disease ◽

Cell Injury ◽

Critical Role ◽

Regulatory Proteins ◽

Specific Cell ◽

Cell Cycle Proteins ◽

Proliferation And Apoptosis ◽

Cell Cycle Regulatory Proteins

The response to glomerular and tubulointerstitial cell injury in most forms of renal disease includes changes in cell number (proliferation and apoptosis) and cell size (hyerptrophy). These events typically precede and may be reponsible for the accumulation of extracellular matrix proteins that leads to a decrease in renal function. There is increasing evidence showing that positive (cyclins and cyclin-dependent kinases) and negative (cyclin-dependent kinase inhibitors) cell cycle regulatory proteins have a critical role in regulating these fundamental cellular responses to immune and nonimmune forms of injury. Data now show that altering specific cell cycle proteins affects renal cell proliferation and improves renal function. Equally exciting is the expanding body of literature showing novel biological roles for cell cycle proteins in the regulation of cell hypertrophy and apoptosis. With increasing understanding of the role for cell cyle regulatory proteins in renal disease comes the hope for potential therapeutic inverventions.

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LPS induces phosphorylation of actin-regulatory proteins leading to actin reassembly and macrophage motility

Journal of Cellular Biochemistry ◽

10.1002/jcb.23330 ◽

2011 ◽

Vol 113 (1) ◽

pp. 80-92 ◽

Cited By ~ 31

Author(s):

Galyna Kleveta ◽

Kinga Borzęcka ◽

Mykola Zdioruk ◽

Maciej Czerkies ◽

Hanna Kuberczyk ◽

...

Keyword(s):

Regulatory Proteins ◽

Actin Regulatory Proteins

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Variants in the VCAM1 gene and risk for symptomatic stroke in sickle cell disease

Blood ◽

10.1182/blood-2001-12-0306 ◽

2002 ◽

Vol 100 (13) ◽

pp. 4303-4309 ◽

Cited By ~ 52

Author(s):

James G. Taylor VI ◽

Delia C. Tang ◽

Sharon A. Savage ◽

Susan F. Leitman ◽

Seth I. Heller ◽

...

Keyword(s):

Sickle Cell ◽

Critical Role ◽

Genome Project ◽

Monogenic Disease ◽

Candidate Snps ◽

Healthy Population ◽

Nucleotide Polymorphisms ◽

Coding Region ◽

A Cell ◽

The Human Genome Project

Stroke is a major cause of morbidity and mortality in sickle cell (SS) disease. Genetic risk factors have been postulated to contribute to this clinical outcome. The human genome project has substantially increased the catalog of variations in genes, many of which could modify the risk for manifestations of disease outcome in a monogenic disease, namely SS. VCAM1 is a cell adhesion molecule postulated to play a critical role in the pathogenesis of SS disease. We identified a total of 33 single nucleotide polymorphisms (SNPs) by sequencing the entire coding region, 2134 bp upstream of the 5′ end of the published cDNA, 217 bp downstream of the 3′ end of the cDNA, and selected intronic regions of the VCAM1 locus. Allelic frequencies for selected SNPs were determined in a healthy population. We subsequently analyzed 4 nonsynonymous coding, 2 synonymous coding, and 4 common promoter SNPs in a genetic association study of clinically apparent stroke in SS disease conducted in a cohort derived from a single institution in Jamaica (51 symptomatic cases and 51 matched controls). Of the 10 candidate SNPs analyzed in this pilot study, the variant allele of the nonsynonymous SNP, VCAM1 G1238C, may be associated with protection from stroke (odds ratio [OR] 0.35, 95% confidence interval [CI] 0.15-0.83, P = .04). Further study is required to confirm the importance of this variant inVCAM1 as a clinically useful modifier of outcome in SS disease.

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Loss of Profilin3 Impairs Spermiogenesis by Affecting Acrosome Biogenesis, Autophagy, Manchette Development and Mitochondrial Organization

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.749559 ◽

2021 ◽

Vol 9 ◽

Author(s):

Naila Umer ◽

Lena Arévalo ◽

Sharang Phadke ◽

Keerthika Lohanadan ◽

Gregor Kirfel ◽

...

Keyword(s):

Actin Polymerization ◽

Regulatory Proteins ◽

Autophagic Flux ◽

Nuclear Fraction ◽

Rna Seq ◽

Head Shape ◽

Male Factor ◽

Protein Levels ◽

Acrosome Formation ◽

Actin Regulatory Proteins

Profilins (PFNs) are key regulatory proteins for the actin polymerization in cells and are encoded in mouse and humans by four Pfn genes. PFNs are involved in cell mobility, cell growth, neurogenesis, and metastasis of tumor cells. The testes-specific PFN3 is localized in the acroplaxome–manchette complex of developing spermatozoa. We demonstrate that PFN3 further localizes in the Golgi complex and proacrosomal vesicles during spermiogenesis, suggesting a role in vesicle transport for acrosome formation. Using CRISPR/Cas9 genome editing, we generated mice deficient for Pfn3. Pfn3–/– males are subfertile, displaying a type II globozoospermia. We revealed that Pfn3–/– sperm display abnormal manchette development leading to an amorphous sperm head shape. Additionally, Pfn3–/– sperm showed reduced sperm motility resulting from flagellum deformities. We show that acrosome biogenesis is impaired starting from the Golgi phase, and mature sperm seems to suffer from a cytoplasm removal defect. An RNA-seq analysis revealed an upregulation of Trim27 and downregulation of Atg2a. As a consequence, mTOR was activated and AMPK was suppressed, resulting in the inhibition of autophagy. This dysregulation of AMPK/mTOR affected the autophagic flux, which is hallmarked by LC3B accumulation and increased SQSTM1 protein levels. Autophagy is involved in proacrosomal vesicle fusion and transport to form the acrosome. We conclude that this disruption leads to the observed malformation of the acrosome. TRIM27 is associated with PFN3 as determined by co-immunoprecipitation from testis extracts. Further, actin-related protein ARPM1 was absent in the nuclear fraction of Pfn3–/– testes and sperm. This suggests that lack of PFN3 leads to destabilization of the PFN3–ARPM1 complex, resulting in the degradation of ARPM1. Interestingly, in the Pfn3–/– testes, we detected increased protein levels of essential actin regulatory proteins, cofilin-1 (CFL1), cofilin-2 (CFL2), and actin depolymerizing factor (ADF). Taken together, our results reveal the importance for PFN3 in male fertility and implicate this protein as a candidate for male factor infertility in humans.

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