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Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other important cellular biology questions. The technologies available to genetically manipulate Dictyostelium cells are well-developed. Transfections can be performed using different selectable markers and marker re-cycling, including homologous recombination and insertional mutagenesis. This is supported by a well-annotated genome. However, these approaches are optimized for axenic cell lines growing in liquid cultures and are difficult to apply to non-axenic wild-type cells, which feed only on bacteria. The mutations that are present in axenic strains disturb Ras signaling, causing excessive macropinocytosis required for feeding, and impair cell migration, which confounds the interpretation of signal transduction and chemotaxis experiments in those strains. Earlier attempts to genetically manipulate non-axenic cells have lacked efficiency and required complex experimental procedures. We have developed a simple transfection protocol that, for the first time, overcomes these limitations. Those series of large improvements to Dictyostelium molecular genetics allow wild-type cells to be manipulated as easily as standard laboratory strains. In addition to the advantages for studying uncorrupted signaling and motility processes, mutants that disrupt macropinocytosis-based growth can now be readily isolated. Furthermore, the entire transfection workflow is greatly accelerated, with recombinant cells that can be generated in days rather than weeks. Another advantage is that molecular genetics can further be performed with freshly isolated wild-type Dictyostelium samples from the environment. This can help to extend the scope of approaches used in these research areas.

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other important cellular biology questions. The technologies available to genetically manipulate Dictyostelium cells are well-developed. Transfections can be performed using different selectable markers and marker re-cycling, including homologous recombination and insertional mutagenesis. This is supported by a well-annotated genome. However, these approaches are optimized for axenic cell lines growing in liquid cultures and are difficult to apply to non-axenic wild-type cells, which feed only on bacteria. The mutations that are present in axenic strains disturb Ras signaling, causing excessive macropinocytosis required for feeding, and impair cell migration, which confounds the interpretation of signal transduction and chemotaxis experiments in those strains. Earlier attempts to genetically manipulate non-axenic cells have lacked efficiency and required complex experimental procedures. We have developed a simple transfection protocol that, for the first time, overcomes these limitations. Those series of large improvements to Dictyostelium molecular genetics allow wild-type cells to be manipulated as easily as standard laboratory strains. In addition to the advantages for studying uncorrupted signaling and motility processes, mutants that disrupt macropinocytosis-based growth can now be readily isolated. Furthermore, the entire transfection workflow is greatly accelerated, with recombinant cells that can be generated in days rather than weeks. Another advantage is that molecular genetics can further be performed with freshly isolated wild-type Dictyostelium samples from the environment. This can help to extend the scope of approaches used in these research areas.

Model organisms have been key to understanding many core biological processes. Dictyostelium amoebae have the attributes required to perform this role for chemotaxis, and by providing an evolutionary distant reference point to mammalian cells, they allow the central features of chemotaxis to be discerned. Here we highlight progress with Dictyostelium in understanding: pseudopod and bleb driven movement; the role of the actin cytoskeleton; chemotactic signal processing, including how cells adapt to background stimulation, and the controversial role of PIP3. Macropinocytosis and the axenic mutations are raised as potential confounding factors, while the identification of new players through proteomics holds great promise.

The cytoskeleton is utilized for a variety of cellular processes, including migration, endocytosis and adhesion. The required molecular components are often shared between different processes, but it is not well understood how the cells balance their use. We find that macropinocytosis and cell migration are negatively correlated. Heavy drinkers move only slowly and vice versa, fast cells do not take big gulps. Both processes are balanced by the lipid phosphatidylinositol 3,4,5-trisphosphate (PIP3). Elevated PIP3 signalling causes a shift towards macropinocytosis and inhibits motility by redirecting the SCAR/WAVE complex, a major nucleator of actin filaments. High resolution microscopy shows that patches with high levels of PIP3 recruit SCAR/WAVE on their periphery, resulting in circular ruffle formation and engulfment. Results shed new light on the role of PIP3, which is commonly thought to promote cell motility.

In eukaryotic chemotaxis, the mechanisms connecting external signals to the motile apparatus remain unclear. The role of the lipid phosphatidylinositol 3,4,5-trisphosphate (PIP3) has been particularly controversial. PIP3 has many cellular roles, notably in growth control and macropinocytosis as well as cell motility. Here we show that PIP3 is not only unnecessary for Dictyostelium discoideum to migrate toward folate, but actively inhibits chemotaxis. We find that macropinosomes, but not pseudopods, in growing cells are dependent on PIP3. PIP3 patches in these cells show no directional bias, and overall only PIP3-free pseudopods orient up-gradient. The pseudopod driver suppressor of cAR mutations (SCAR)/WASP and verprolin homologue (WAVE) is not recruited to the center of PIP3 patches, just the edges, where it causes macropinosome formation. Wild-type cells, unlike the widely used axenic mutants, show little macropinocytosis and few large PIP3 patches, but migrate more efficiently toward folate. Tellingly, folate chemotaxis in axenic cells is rescued by knocking out phosphatidylinositide 3-kinases (PI 3-kinases). Thus PIP3 promotes macropinocytosis and interferes with pseudopod orientation during chemotaxis of growing cells.

Summary Background SCAR/WAVE is a principal regulator of pseudopod growth in crawling cells. It exists in a stable pentameric complex, which is regulated at multiple levels that are only beginning to be understood. SCAR/WAVE is phosphorylated at multiple sites, but how this affects its biological activity is unclear. Here we show that dephosphorylation of Dictyostelium SCAR controls normal pseudopod dynamics. Results We demonstrate that the C-terminal acidic domain of most Dictyostelium SCAR is basally phosphorylated at four serine residues. A small amount of singly phosphorylated SCAR is also found. SCAR phosphorylation site mutants cannot replace SCAR's role in the pseudopod cycle, though they rescue cell size and growth. Unphosphorylatable SCAR is hyperactive—excessive recruitment to the front results in large pseudopods that fail to bifurcate because they continually grow forward. Conversely, phosphomimetic SCAR is weakly active, causing frequent small, disorganized pseudopods. Even in its regulatory complex, SCAR is normally held inactive by an interaction between the phosphorylated acidic and basic domains. Loss of basic residues complementary to the acidic phosphosites yields a hyperactive protein similar to unphosphorylatable SCAR. Conclusions Regulated dephosphorylation of a fraction of the cellular SCAR pool is a key step in SCAR activation during pseudopod growth. Phosphorylation increases autoinhibition of the intact complex. Dephosphorylation weakens this interaction and facilitates SCAR activation but also destabilizes the protein. We show that SCAR is specifically dephosphorylated in pseudopods, increasing activation by Rac and lipids and supporting positive feedback of pseudopod growth.

The ability to respond and adapt to changes in the physical environment is a universal and essential cellular property. Here we demonstrated that cells respond to mechanical compressive stress by rapidly inducing autophagosome formation. We measured this response in both Dictyostelium and mammalian cells, indicating that this is an evolutionarily conserved, general response to mechanical stress. In Dictyostelium, the number of autophagosomes increased 20-fold within 10 min of 1 kPa pressure being applied and a similar response was seen in mammalian cells after 30 min. We showed in both cell types that autophagy is highly sensitive to changes in mechanical pressure and the response is graduated, with half-maximal responses at ~0.2 kPa, similar to other mechano-sensitive responses. We further showed that the mechanical induction of autophagy is TOR-independent and transient, lasting until the cells adapt to their new environment and recover their shape. The autophagic response is therefore part of an integrated response to mechanical challenge, allowing cells to cope with a continuously changing physical environment.

I-BAR (inverse-Bin/amphiphysin/Rvs)-domain-containing proteins such as IRSp53 (insulin receptor substrate of 53 kDa) associate with outwardly curved membranes and connect them to proteins involved in actin dynamics. Research on I-BAR proteins has focussed on possible roles in filopod and lamellipod formation, but their full physiological function remains unclear. The social amoeba Dictyostelium encodes a single I-BAR/SH3 (where SH3 is Src homology 3) protein, called IBARa, along with homologues of proteins that interact with IRSp53 family proteins in mammalian cells, providing an excellent model to study its cellular function. Disruption of the gene encoding IBARa leads to a mild defect in development, but filopod and pseudopod dynamics are unaffected. Furthermore, ectopically expressed IBARa does not induce filopod formation and does not localize to filopods. Instead, IBARa associates with clathrin puncta immediately before they are endocytosed. This role is conserved: human BAIAP2L2 (brain-specific angiogenesis inhibitor 1-associated protein 2-like 2) also tightly co-localizes with clathrin plaques, although its homologues IRSp53 and IRTKS (insulin receptor tyrosine kinase substrate) associate with other punctate structures. The results from the present study suggest that I-BAR-containing proteins help generate the mem-brane curvature required for endocytosis and implies an unexpected role for IRSp53 family proteins in vesicle trafficking.

Dictyostelium has a mature technology for molecular-genetic manipulation based around transfection using several different selectable markers, marker re-cycling, homologous recombination and insertional mutagenesis, all supported by a well-annotated genome. However this technology is optimized for mutant, axenic cells that, unlike non-axenic wild type, can grow in liquid medium. There is a pressing need for methods to manipulate wild type cells and ones with defects in macropinocytosis, neither of which can grow in liquid media. Here we present a panel of molecular genetic techniques based on the selection of Dictyostelium transfectants by growth on bacteria rather than liquid media. As well as extending the range of strains that can be manipulated, these techniques are faster than conventional methods, often giving usable numbers of transfected cells within a few days. The methods and plasmids described here allow efficient transfection with extrachromosomal vectors, as well as chromosomal integration at a 'safe haven' for relatively uniform cell-to-cell expression, efficient gene knock-in and knock-out and an inducible expression system. We have thus created a complete new system for the genetic manipulation of Dictyostelium cells that no longer requires cell feeding on liquid media.

Chemotaxis, or directional movement toward extracellular chemical gradients, is an important property of cells that is mediated through G-protein-coupled receptors (GPCRs). Although many chemotaxis pathways downstream of G beta gamma have been identified, few G alpha effectors are known. G alpha effectors are of particular importance because they allow the cell to distinguish signals downstream of distinct chemoattractant GPCRs. Here we identify GflB, a G alpha 2 binding partner that directly couples the Dictyostelium cyclic AMP GPCR to Rap1. GflB localizes to the leading edge and functions as a G alpha-stimulated, Rap1-specific guanine nucleotide exchange factor required to balance Ras and Rap signaling. The kinetics of GflB translocation are fine-tuned by GSK-3 phosphorylation. Cells lacking GflB display impaired Rap1/Ras signaling and actin and myosin dynamics, resulting in defective chemotaxis. Our observations demonstrate that GflB is an essential upstream regulator of chemoattractant-mediated cell polarity and cytoskeletal reorganization functioning to directly link G alpha activation to monomeric G-protein signaling.

Melanoma cells break down lysophosphatidic acid from the environment, creating a chemotactic gradient that the tumor cells then follow; this provides an explanation for the rapid metastasis of melanoma., The high mortality of melanoma is caused by rapid spread of cancer cells, which occurs unusually early in tumour evolution. Unlike most solid tumours, thickness rather than cytological markers or differentiation is the best guide to metastatic potential. Multiple stimuli that drive melanoma cell migration have been described, but it is not clear which are responsible for invasion, nor if chemotactic gradients exist in real tumours. In a chamber-based assay for melanoma dispersal, we find that cells migrate efficiently away from one another, even in initially homogeneous medium. This dispersal is driven by positive chemotaxis rather than chemorepulsion or contact inhibition. The principal chemoattractant, unexpectedly active across all tumour stages, is the lipid agonist lysophosphatidic acid (LPA) acting through the LPA receptor LPAR1. LPA induces chemotaxis of remarkable accuracy, and is both necessary and sufficient for chemotaxis and invasion in 2-D and 3-D assays. Growth factors, often described as tumour attractants, cause negligible chemotaxis themselves, but potentiate chemotaxis to LPA. Cells rapidly break down LPA present at substantial levels in culture medium and normal skin to generate outward-facing gradients. We measure LPA gradients across the margins of melanomas in vivo, confirming the physiological importance of our results. We conclude that LPA chemotaxis provides a strong drive for melanoma cells to invade outwards. Cells create their own gradients by acting as a sink, breaking down locally present LPA, and thus forming a gradient that is low in the tumour and high in the surrounding areas. The key step is not acquisition of sensitivity to the chemoattractant, but rather the tumour growing to break down enough LPA to form a gradient. Thus the stimulus that drives cell dispersal is not the presence of LPA itself, but the self-generated, outward-directed gradient., Author Summary Melanoma is feared because it spreads very rapidly when tumours are relatively small. It is not known why this metastasis is so efficient and aggressive. In particular, it is not known what drives melanoma cells to start to migrate out from the tumour. Here, we have studied the chemical signals that guide the migration of melanoma cells. We find that a component of serum, lysophosphatidic acid (LPA), functions as a remarkably strong attractant for all of the melanoma cells that we examined. We also observe that melanoma cells rapidly break down LPA. We conclude that melanomas create their own gradients of LPA, with low LPA in the tumour and high LPA outside. Since melanoma cells are attracted by LPA, this LPA gradient around the melanomas serves as a signal that drives the tumour cells out into the surrounding skin and blood vessels. Finally, we show that such gradients exist in a mouse model of melanoma. Self-generated LPA gradients are therefore an intriguing new driver for melanoma dispersal.

In growing Dictyostelium discoideum, PIP3 is unnecessary for migration toward folate and actively inhibits chemotaxis and pseudopod formation by promoting macropinocytosis., In eukaryotic chemotaxis, the mechanisms connecting external signals to the motile apparatus remain unclear. The role of the lipid phosphatidylinositol 3,4,5-trisphosphate (PIP3) has been particularly controversial. PIP3 has many cellular roles, notably in growth control and macropinocytosis as well as cell motility. Here we show that PIP3 is not only unnecessary for Dictyostelium discoideum to migrate toward folate, but actively inhibits chemotaxis. We find that macropinosomes, but not pseudopods, in growing cells are dependent on PIP3. PIP3 patches in these cells show no directional bias, and overall only PIP3-free pseudopods orient up-gradient. The pseudopod driver suppressor of cAR mutations (SCAR)/WASP and verprolin homologue (WAVE) is not recruited to the center of PIP3 patches, just the edges, where it causes macropinosome formation. Wild-type cells, unlike the widely used axenic mutants, show little macropinocytosis and few large PIP3 patches, but migrate more efficiently toward folate. Tellingly, folate chemotaxis in axenic cells is rescued by knocking out phosphatidylinositide 3-kinases (PI 3-kinases). Thus PIP3 promotes macropinocytosis and interferes with pseudopod orientation during chemotaxis of growing cells.

Inducible expression systems are very convenient for proteins that induce strong side effects such as retardation of growth or development and are essential for the expression of toxic proteins. In this chapter we describe the doxycycline-inducible expression system, optimized for the controlled expression in. Two types of inducible plasmids are presented, in which transcription is induced by either adding or removing doxycycline, respectively. Detailed protocols are provided for the construction of the plasmids and the inducible expression of the target protein.

Direct visualization chambers are considered the gold standard for measuring and analyzing chemotactic responses, because they allow detailed analysis of cellular behavior during the process of chemotaxis. We have previously described the Insall chamber, an improved chamber for measuring cancer cell chemotaxis. Here, we describe in detail how this system can be used to perform two key assays for both fast- and slow-moving mammalian and nonmammalian cell types. This allows for the detailed analysis of chemotactic responses in linear gradients at the levels of both overall cell behavior and subcellular dynamics.

Inducible expression systems are very convenient for proteins that induce strong side effects such as retardation of growth or development and are essential for the expression of toxic proteins. In this chapter we describe the doxycycline-inducible expression system, optimized for the controlled expression in. Two types of inducible plasmids are presented, in which transcription is induced by either adding or removing doxycycline, respectively. Detailed protocols are provided for the construction of the plasmids and the inducible expression of the target protein.

Cells use their cytoskeletons to move, polarise, divide and maintain organisation within multicellular tissues. Actin is a highly conserved essential building block of the cytoskeleton that forms cables and struts, which are constantly remodelled by more than 100 different actin-binding proteins.

Dock (dedicator of cytokinesis) proteins represent a family of guanine nucleotide exchange factors (GEFs) that include the well-studied Dock180 family and the poorly characterised zizimin family. Our current understanding of Dock180 function is that it regulates Rho small GTPases and thus has a role in a number of cell processes, including cell migration, development and division. Here, we use a tractable model for cell motility research, Dictyostelium discoideum, to help elucidate the role of the related zizimin proteins. We show that gene ablation of zizA causes no change in development, whereas ablation of zizB gives rise to an aberrant developmental morphology and a reduction in cell directionality and velocity, and altered cell shape. Fluorescently labelled ZizA protein associates with the microtubule-organising centre (MTOC), whereas ZizB is enriched in the cortex. Overexpression of ZizB also causes an increase in the number of filopodia and a partial inhibition of cytokinesis. Analysis of ZizB protein binding partners shows that it interacts with Rac1a and a range of actin-associated proteins. In conclusion, our work provides insight into the molecular and cellular functions of zizimin GEF proteins, which are shown to have a role in cell movement, filopodia formation and cytokinesis.

Dock (Dedicator of Cytokinesis) proteins represent a family of Guanine nucleotide Exchange Factors (GEFs) that include the well studied Dock180 family and the poorly characterised zizimin family. Our current understanding of Dock180 function is to regulate Rho small GTPases, playing a role in a number of cell processes including cell migration, development and division. Here, we have employed a tractable model for cell motility research, Dictyostelium discoideum, to help elucidate the role of the related zizimin proteins. We show that gene ablation of zizA causes no change in development whereas ablation of zizB gives rise to an aberrant developmental morphology and a reduction in cell directionality and velocity, and altered cell shape. Fluorescently labeled ZizA protein associates with the microtubule organizing centre (MTOC), whereas the ZizB protein exhibits cortical enrichment. Overexpression of ZizB also causes an increase in the number filopodia and a partial inhibition of cytokinesis. Analysis of ZizB protein binding partners indicates interacts with Rac1a and a range of actin-interacting proteins. In conclusion our work provides the first insight into the molecular and cellular functions of zizimin GEF proteins playing a role in cell movement, filopodia formation and cytokinesis.

The WASP family control formation of actin filaments through the Arp2/3 complex. Subfamiles include WASP, SCAR/WAVE, WASH, and WHAMM. We show that the family is unexpectedly ancient and that all subfamilies are now identified. This work also identifies a subfamily-specific control mechanism, and an emerging bias towards vesicular roles of actin., WASP family proteins control actin polymerization by activating the Arp2/3 complex. Several subfamilies exist, but their regulation and physiological roles are not well understood, nor is it even known if all subfamilies have been identified. Our extensive search reveals few novel WASP family proteins. The WASP, WASH, and SCAR/WAVE subfamilies are evolutionarily ancient, with WASH the most universally present, whereas WHAMM/JMY first appears in invertebrates. An unusual Dictyostelium WASP homologue that has lost the WH1 domain has retained its function in clathrin-mediated endocytosis, demonstrating that WASPs can function with a remarkably diverse domain topology. The WASH and SCAR/WAVE regulatory complexes are much more rigidly maintained; their domain topology is highly conserved, and all subunits are present or lost together, showing that the complexes are ancient and functionally interdependent. Finally, each subfamily has a distinctive C motif, indicating that this motif plays a specific role in each subfamily's function, unlike the generic V and A motifs. Our analysis identifies which features are universally conserved, and thus essential, and which are branch-specific modifications. It also shows the WASP family is more widespread and diverse than currently appreciated and unexpectedly biases the physiological role of the Arp2/3 complex toward vesicle traffic.

Cell division requires the tight coordination of multiple cytoskeletal pathways. The best understood of these involves myosin-II-dependent constriction around the cell equator, but both Dictyostelium and mammalian cells also use a parallel, adhesion-dependent mechanism to generate furrows. We show that the actin nucleation factor SCAR/WAVE is strongly activated during Dictyostelium cytokinesis. This activation localises to large polar protrusions, driving separation of the daughter cells. This continues for 10 minutes after division before the daughter cells revert to normal random motility, indicating that this is a tightly regulated process. We demonstrate that SCAR activity is essential to drive myosin-II-independent cytokinesis, and stabilises the furrow, ensuring symmetrical division. SCAR is also responsible for the generation of MiDASes, mitosis-specific actin-rich adhesions. Loss of SCAR in both Dictyostelium and Drosophila leads to a similar mitotic phenotype, with severe mitotic blebbing, indicating conserved functionality. We also find that the microtubule end-binding protein EB1 is required to restrict SCAR localisation and direct migration. EB1-null cells also exhibit decreased adhesion during mitosis. Our data reveal a spindle-directed signalling pathway that regulates SCAR activity, migration and adhesion at mitosis.

During chemotaxis, phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ) accumulates at the leading edge of a eukaryotic cell, where it induces the formation of pseudopodia. PIP 3 has been suggested to be the compass of cells navigating in gradients of signaling molecules. Recent observations suggest that chemotaxis is more complex than previously anticipated. Complete inhibition of all PIP 3 signaling has little effect, and alternative pathways have been identified. In addition, selective pseudopod growth and retraction are more important in directing cell movement than is the place where new pseudopodia are formed.

cGMP is used as a second messenger in many eukaryotes. cGMP signaling requires at least three components: Guanylyl cyclases synthesize cGMP from GTP. Specific cGMP-binding proteins propagate the signal, usually by phosphorylation of their target proteins. Finally, phosphodiesterases terminate the cGMP signal by hydrolyzing cGMP to 5'cGMP. Recently, all guanylyl cyclases and most of the cGMP target proteins and phosphodiesterases of the cellular slime mold Dictyostelium discoideum have been identified. Characterization of these enzymes show them to be structurally and evolutionarily distinct from their bacterial and metazoan counterparts. In this chapter we review the properties of the Dictyostelium guanylyl cyclases and discuss their role in the unusual cGMP pathway of Dictyostelium.

cGMP is used as a second messenger in many eukaryotes. cGMP signaling requires at least three components: Guanylyl cyclases synthesize cGMP from GTP. Specific cGMP-binding proteins propagate the signal, usually by phosphorylation of their target proteins. Finally, phosphodiesterases terminate the cGMP signal by hydrolyzing cGMP to 5′cGMP. Recently, all guanylyl cyclases and most of the cGMP target proteins and phosphodiesterases of the cellular slime mold Dictyostelium discoideum have been identified. Characterization of these enzymes show them to be structurally and evolutionarily distinct from their bacterial and metazoan counterparts. In this chapter we review the properties of the Dictyostelium guanylyl cyclases and discuss their role in the unusual cGMP pathway of Dictyostelium .

Dock (dedicator of cytokinesis) proteins represent a family of guanine nucleotide exchange factors (GEFs) that include the well-studied Dock180 family and the poorly characterised zizimin family. Our current understanding of Dock180 function is that it regulates Rho small GTPases and thus has a role in a number of cell processes, including cell migration, development and division. Here, we use a tractable model for cell motility research, Dictyostelium discoideum, to help elucidate the role of the related zizimin proteins. We show that gene ablation of zizA causes no change in development, whereas ablation of zizB gives rise to an aberrant developmental morphology and a reduction in cell directionality and velocity, and altered cell shape. Fluorescently labelled ZizA protein associates with the microtubule-organising centre (MTOC), whereas ZizB is enriched in the cortex. Overexpression of ZizB also causes an increase in the number of filopodia and a partial inhibition of cytokinesis. Analysis of ZizB protein binding partners shows that it interacts with Rac1a and a range of actin-associated proteins. In conclusion, our work provides insight into the molecular and cellular functions of zizimin GEF proteins, which are shown to have a role in cell movement, filopodia formation and cytokinesis.

There has been a growing appreciation over the last decade that chemotaxis plays an important role in cancer migration, invasion and metastasis. Research into the field of cancer cell chemotaxis is still in its infancy and traditional investigative tools have been developed with other cell types and purposes in mind. Direct visualisation chambers are considered the gold standard for investigating the behaviour of cells migrating in a chemotactic gradient. We therefore drew up a list of key attributes that a chemotaxis chamber should have for investigating cancer cell chemotaxis. These include (1) compatibility with thin cover slips for optimal optical properties and to allow use of high numerical aperture (NA) oil immersion objectives; (2) gradients that are relatively stable for at least 24 hours due to the slow migration of cancer cells; (3) gradients of different steepnesses in a single experiment, with defined, consistent directions to avoid the need for complicated analysis; and (4) simple handling and disposability for use with medical samples. Here we describe and characterise the Insall chamber, a novel direct visualisation chamber. We use it to show GFP-lifeact transfected MV3 melanoma cells chemotaxing using a 60x high NA oil immersion objective, which cannot usually be done with other chemotaxis chambers. Linear gradients gave very efficient chemotaxis, contradicting earlier results suggesting that only polynomial gradients were effective. In conclusion, the chamber satisfies our design criteria, most importantly allowing high NA oil immersion microscopy to track chemotaxing cancer cells in detail over 24 hours.