71 research outputs found
Cells assemble invadopodia-like structures and invade into matrigel in a matrix metalloprotease dependent manner in the circular invasion assay
The ability of tumor cells to invade is one of the hallmarks of the metastatic phenotype. To elucidate the mechanisms by which tumor cells acquire an invasive phenotype, in vitro assays have been developed that mimic the process of cancer cell invasion through basement membrane or in the stroma. We have extended the characterization of the circular invasion assay and found that it provides a simple and amenable system to study cell invasion in matrix in an environment that closely mimics 3D invasion. Furthermore, it allows detailed microscopic analysis of both live and fixed cells during the invasion process. We find that cells invade in a protease dependent manner in this assay and that they assemble focal adhesions and invadopodia that resemble structures visualized in 3D embedded cells. We propose that this is a useful assay for routine and medium throughput analysis of invasion of cancer cells in vitro and the study of cells migrating in a 3D environment
Recommended from our members
The WAVE Regulatory Complex Is Required to Balance Protrusion and Adhesion in Migration
YesCells migrating over 2D substrates are required to polymerise actin at the leading edge to form lamellipodia protrusions and nascent adhesions to anchor the protrusion to the substrate. The major actin nucleator in lamellipodia formation is the Arp2/3 complex, which is activated by the WAVE regulatory complex (WRC). Using inducible Nckap1 floxed mouse embryonic fibroblasts (MEFs), we confirm that the WRC is required for lamellipodia formation, and importantly, for generating the retrograde flow of actin from the leading cell edge. The loss of NCKAP1 also affects cell spreading and focal adhesion dynamics. In the absence of lamellipodium, cells can become elongated and move with a single thin pseudopod, which appears devoid of N-WASP. This phenotype was more prevalent on collagen than fibronectin, where we observed an increase in migratory speed. Thus, 2D cell migration on collagen is less dependent on branched actin
A unique talin homologue with a villin headpiece-like domain is required for multicellular morphogenesis in Dictyostelium
AbstractMolecules involved in the interaction between the extracellular matrix, cell membrane and cytoskeleton are of central importance in morphogenesis. Talin is a large cytoskeletal protein with a modular structure consisting of an amino-terminal membrane-interacting domain, with sequence similarities to members of the band 4.1 family, and a carboxy-terminal region containing F-actin-binding and vinculin-binding domains [1,2]. It also interacts with the cytoplasmic tail of ÎČ integrins which, on the external face of the membrane, bind to extracellular matrix proteins [3]. The possible roles of talin in multicellular morphogenesis in development remain largely unexplored. In Dictyostelium, a eukaryotic microorganism capable of multicellular morphogenesis, a talin homologue (TALA) has previously been identified and shown to play an important role in cell-to-substrate adhesion and maintenance of normal elastic properties of the cell [4â6]. Here, we describe a second talin homologue (TALB) that is required for multicellular morphogenesis in the development of Dictyostelium. Unlike any other talin characterised to date, it contains an additional carboxy-terminal domain homologous to the villin headpiece
WASP restricts active Rac to maintain cells' front-rear polarization
YesEfficient motility requires polarized cells, with pseudopods at the front and a retracting rear. Polarization is maintained by restricting the pseudopod catalyst, active Rac, to the front. Here, we show that the actin nucleation-promoting factor Wiskott-Aldrich syndrome protein (WASP) contributes to maintenance of front-rear polarity by controlling localization and cellular levels of active Rac. Dictyostelium cells lacking WASP inappropriately activate Rac at the rear, which affects their polarity and speed. WASPâs Cdc42 and Rac interacting binding (âCRIBâ) motif has been thought to be essential for its activation. However, we show that the CRIB motifâs biological role is unexpectedly complex. WASP CRIB mutants are no longer able to restrict Rac activity to the front, and cannot generate new pseudopods when SCAR/WAVE is absent. Overall levels of Rac activity also increase when WASP is unable to bind to Rac. However, WASP without a functional CRIB domain localizes normally at clathrin pits during endocytosis, and activates Arp2/3 complex. Similarly, chemical inhibition of Rac does not affect WASP localization or activation at sites of endocytosis. Thus, the interaction between small GTPases and WASP is more complex than previously thoughtâRac regulates a subset of WASP functions, but WASP reciprocally restricts active Rac through its CRIB motif.Cancer Research UK grants A15672, A24450, and multidisciplinary grant A20017
Curved Tails in Polymerization-Based Bacterial Motility
The curved actin ``comet-tail'' of the bacterium Listeria monocytogenes is a
visually striking signature of actin polymerization-based motility. Similar
actin tails are associated with Shigella flexneri, spotted-fever Rickettsiae,
the Vaccinia virus, and vesicles and microspheres in related in vitro systems.
We show that the torque required to produce the curvature in the tail can arise
from randomly placed actin filaments pushing the bacterium or particle. We find
that the curvature magnitude determines the number of actively pushing
filaments, independent of viscosity and of the molecular details of force
generation. The variation of the curvature with time can be used to infer the
dynamics of actin filaments at the bacterial surface.Comment: 8 pages, 2 figures, Latex2
Atomic Force Microscopy of height fluctuations of fibroblast cells
We investigated the nanometer scale height fluctuations of 3T3 fibroblast
cells with the atomic force microscope (AFM) under physiological conditions.
Correlation between these fluctuations and lateral cellular motility can be
observed. Fluctuations measured on leading edges appear to be predominantly
related to actin polymerization-depolymerization processes. We found fast (5
Hz) pulsatory behavior with 1--2 nm amplitude on a cell with low motility
showing emphasized structure of stress fibres. Myosin driven contractions of
stress fibres are thought to induce this pulsation.Comment: 6 pages, 5 figures, 1 tabl
Recommended from our members
Genetically engineered multicistronic allele of Pmel yielding highly specific CreERT2-mediated recombination in the melanocyte lineage
YesGenetic approaches that allow lineage tracing are essential to our future understanding of melanocytes and melanoma. To date, the approaches used to label melanocytes in mice have relied on random integration of transgenes driven by the promoters of the Tyrosinase and Dopachrome tautomerase genes, knock-in to the Dopachrome tautomerase locus or knock-in to the Mlana locus in a bacterial artificial chromosome. These strategies result in expression in other tissues such as telencephalon and other cell types such as nerves. Here we used homologous recombination in mouse embryonic stem cells to generate a targeted multicistronic allele of the Pmel locus that drives melanocyte-specific expression of CreERT2, nuclear localised H2B-Cerulean and membrane localised marcks-mKate2 allowing live imaging of melanocytes and activation of other conditional alleles. We combined this allele with R26R-EYFP mice allowing induction of EYFP expression on administration of tamoxifen or its metabolite 4-OHT. The fluorescent proteins H2B-Cerulean and marcks-mKate2 label the cell nucleus and plasma membrane respectively allowing live imaging and FACS isolation of melanoblasts and melanocytes as well as serving to provide an internal control allowing estimation of recombination efficiency after administration of tamoxifen. We demonstrate the utility of the transgene in embryonic and adult tissues.Cancer Research UK. Grant Number: A15673. Medical Research Council. Grant Number: MC_PC_U127527200. National Centre for the Replacement Refinement and Reduction of Animals in Research. Grant Number: NC/K001612/1. North West Cancer Research Fund. Grant Number: CR1132. Medical Research Scotland. Grant Number: 436FR
Actin-Based Protrusions: Promoters or Inhibitors of Cancer Invasion?
In a recent issue of Cell, Silva and colleagues reported the identification of CYFIP1, a member of the actin-assembly-promoting Scar/WAVE complex, as an invasion suppressor in epithelial cancers. This study challenges ideas about the role of actin in cancer invasion
- âŠ