Metastasis is responsible for the greatest number of cancer deaths. Metastatic disease, or the movement of cancer cells from one site to another, is a complex process requiring dramatic remodelling of the cell cytoskeleton. The various components of the cytoskeleton, actin (microfilaments), microtubules (MTs) and intermediate filaments, are highly integrated and their functions are well orchestrated in normal cells. In contrast, mutations and abnormal expression of cytoskeletal and cytoskeletal-associated proteins play an important role in the ability of cancer cells to resist chemotherapy and metastasize. Studies on the role of actin and its interacting partners have highlighted key signalling pathways, such as the Rho GTPases, and downstream effector proteins that, through the cytoskeleton, mediate tumour cell migration, invasion and metastasis. An emerging role for MTs in tumour cell metastasis is being unravelled and there is increasing interest in the crosstalk between key MT interacting proteins and the actin cytoskeleton, which may provide novel treatment avenues for metastatic disease. Improved understanding of how the cytoskeleton and its interacting partners influence tumour cell migration and metastasis has led to the development of novel therapeutics against aggressive and metastatic disease. LINKED ARTICLESThis article is part of a themed section on Cytoskeleton, Extracellular Matrix, Cell Migration, Wound Healing and Related Topics. To view the other articles in this section visit http://dx.doi. org/10.1111/bph.2014.171.issue-24 Abbreviations ABP, actin-binding protein; EB1, end-binding protein 1; ECM, extracellular matrix; FA, focal adhesion; F-actin, filamentous actin; FC, focal contact; GEF, guanine nucleotide exchange factor; G-actin, monomeric actin; IF, intermediate filament; LIMK, LIM kinase; MLC, myosin light chain; MT, microtubule; MTOC, microtubule-organizing centre; ROCK, Rho-associated coiled-coil forming protein kinase; Tm, tropomyosin; Tm5NM1, tropomyosin 5 non-muscle isoform 1 IntroductionMetastasis accounts for the vast majority of cancer deaths (Schroeder et al., 2012) and is the result of movement of cancer cells from the primary site (site of origin of cancer) to a distant site or organ. The metastatic spread of cancer cells is a highly selective process consisting of a series of discrete, sequential steps, which have been modelled into a 'metastatic cascade'. To generate metastatic lesions, tumour cells must successfully complete all steps of this process: detachment from the primary tumour → cell migration and invasion → intravasation -transport through vessels and anoikis (cell detachment-induced cell death) evasion -extravasation → growth of secondary tumour (Figure 1; reviewed in Steeg, 2006). Throughout this complex process, cell movements and alterations in cell shape require dramatic spatial and temporal reorganization of the cell cytoskeleton. The three major cytoskeletal filaments are the microfilaments (actin), intermediate filaments (IFs) and microtubules (MTs), an...

HIGHLIGHTS3D bioprinter for the highthroughput production of embedded 3D multicellular spheroids Bioprinted spheroids are biologically similar to manually produced spheroids Parameters such as spheroid size and embedding affect the response toward doxorubicin High-throughput screening applications with high content imaging and viability assay

Maintenance of an optimal redox environment is critical for appropriate functioning of cellular processes and cell survival. Despite the importance of maintaining redox homeostasis, it is not clear how the optimal redox potential is sensed and set, and the processes that impact redox on a cellular/organellar level are poorly understood. The genetic bases of cellular redox homeostasis were investigated using a green fluorescent protein (GFP) based redox probe, roGFP2 and a pH sensitive GFP-based probe, pHluorin. The use of roGFP2, in conjunction with pHluorin, enabled determination of pH-adjusted sub-cellular redox potential in a non-invasive and real-time manner. A genome-wide screen using both the non-essential and essential gene collections was carried out in Saccharomyces cerevisiae using cytosolic-roGFP2 to identify factors essential for maintenance of cytosolic redox state under steady-state conditions. 102 genes of diverse function were identified that are required for maintenance of cytosolic redox state. Mutations in these genes led to shifts in the half-cell glutathione redox potential by 75-10 mV. Interestingly, some specific oxidative stress-response processes were identified as over-represented in the data set. Further investigation of the role of oxidative stress-responsive systems in sub-cellular redox homeostasis was conducted using roGFP2 constructs targeted to the mitochondrial matrix and peroxisome and E GSH was measured in cells in exponential and stationary phase. Analyses allowed for the identification of key redox systems on a sub-cellular level and the identification of novel genes involved in the regulation of cellular redox homeostasis.

T cell receptor (TCR) triggering by antigen results in metabolic reprogramming that, in turn, facilitates T cells' exit from quiescence. The increased nutrient requirements of activated lymphocytes are met in part by upregulation of cell surface transporters and enhanced uptake of amino acids, fatty acids and glucose from the environment. However, the role of intracellular pathways of amino acid biosynthesis in T cell activation is relatively unexplored. Asparagine (Asn) is a non-essential amino acid that can be synthesized intracellularly through the glutamine-hydrolyzing enzyme asparagine synthetase (ASNS). We set out to define the requirements for uptake of extracellular Asn and ASNS activity in CD8 + T cell activation. At early timepoints of activation in vitro, CD8 + T cells expressed little or no ASNS and, as a consequence, viability and TCR-stimulated growth, activation and metabolic reprogramming were substantially impaired under conditions of Asn deprivation. At later timepoints (>24h of activation), TCR-induced mTOR-dependent signals resulted in upregulation of ASNS, that endowed CD8 + T cells with the capacity to function independently of extracellular Asn. Thus, our data suggest that the coordinated upregulation of ASNS expression and uptake of extracellular Asn is involved in optimal T cell effector responses.