Discrepancies in indel software resolution with somatic CRISPR/Cas9 tumorigenesis models.

CRISPR/Cas9 gene editing has evolved from a simple laboratory tool to a powerful method of in vivo genomic engineering. As the applications of CRISPR/Cas9 technology have grown, the need to characterize the breadth and depth of indels generated by editing has expanded. Traditionally, investigators use one of several publicly-available platforms to determine CRISPR/Cas9-induced indels in an edited sample. However, to our knowledge, there has not been a cross-platform comparison of available indel analysis software in samples generated from somatic in vivo mouse models. Our group has pioneered using CRISPR/Cas9 to generate somatic primary mouse models of malignant peripheral nerve sheath tumors (MPNSTs) through genetic editing of Nf1. Here, we used sequencing data from the in vivo editing of the Nf1 gene in our CRISPR/Cas9 tumorigenesis model to directly compare results across four different software platforms. By analyzing the same genetic target across a wide panel of cell lines with the same sequence file, we are able to draw systematic conclusions about the differences in these software programs for analysis of in vivo-generated indels. Surprisingly, we report high variability in the reported number, size, and frequency of indels across each software platform. These data highlight the importance of selecting indel analysis platforms specific to the context that the gene editing approach is being applied. Taken together, this analysis shows that different software platforms can report widely divergent indel data from the same sample, particularly if larger indels are present, which are common in somatic, in vivo CRISPR/Cas9 tumor models.

CDK4/6-MEK Inhibition in MPNSTs Causes Plasma Cell Infiltration, Sensitization to PD-L1 Blockade, and Tumor Regression.

PURPOSE: Malignant peripheral nerve sheath tumors (MPNST) are lethal, Ras-driven sarcomas that lack effective therapies. We investigated effects of targeting cyclin-dependent kinases 4 and 6 (CDK4/6), MEK, and/or programmed death-ligand 1 (PD-L1) in preclinical MPNST models. EXPERIMENTAL DESIGN: Patient-matched MPNSTs and precursor lesions were examined by FISH, RNA sequencing, IHC, and Connectivity-Map analyses. Antitumor activity of CDK4/6 and MEK inhibitors was measured in MPNST cell lines, patient-derived xenografts (PDX), and de novo mouse MPNSTs, with the latter used to determine anti-PD-L1 response. RESULTS: Patient tumor analyses identified CDK4/6 and MEK as actionable targets for MPNST therapy. Low-dose combinations of CDK4/6 and MEK inhibitors synergistically reactivated the retinoblastoma (RB1) tumor suppressor, induced cell death, and decreased clonogenic survival of MPNST cells. In immune-deficient mice, dual CDK4/6-MEK inhibition slowed tumor growth in 4 of 5 MPNST PDXs. In immunocompetent mice, combination therapy of de novo MPNSTs caused tumor regression, delayed resistant tumor outgrowth, and improved survival relative to monotherapies. Drug-sensitive tumors that regressed contained plasma cells and increased cytotoxic T cells, whereas drug-resistant tumors adopted an immunosuppressive microenvironment with elevated MHC II-low macrophages and increased tumor cell PD-L1 expression. Excitingly, CDK4/6-MEK inhibition sensitized MPNSTs to anti-PD-L1 immune checkpoint blockade (ICB) with some mice showing complete tumor regression. CONCLUSIONS: CDK4/6-MEK inhibition induces a novel plasma cell-associated immune response and extended antitumor activity in MPNSTs, which dramatically enhances anti-PD-L1 therapy. These preclinical findings provide strong rationale for clinical translation of CDK4/6-MEK-ICB targeted therapies in MPNST as they may yield sustained antitumor responses and improved patient outcomes.

Loss of Nf1 and Ink4a/Arf Are Associated with Sex-Dependent Growth Differences in a Mouse Model of Embryonal Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is an aggressive form of cancer that accounts for half of all pediatric soft tissue sarcomas. Little progress has been made in improving survival outcomes over the past three decades. Mouse models of rhabdomyosarcoma are a critical component of translational research aimed at understanding tumor biology and developing new, improved therapies. Though several models exist, many common mutations found in human rhabdomyosarcoma tumors remain unmodeled and understudied. This study describes a new model of embryonal rhabdomyosarcoma driven by the loss of Nf1 and Ink4a/Arf, two mutations commonly found in patient tumors. We find that this new model is histologically similar to other previously-published rhabdomyosarcoma models, although it substantially differs in the time required for tumor onset and in tumor growth kinetics. We also observe unique sex-dependent phenotypes in both primary and newly-developed orthotopic syngeneic allograft tumors that are not present in previous models. Using in vitro and in vivo studies, we examined the response to vincristine, a component of the standard-of-care chemotherapy for RMS. The findings from this study provide valuable insight into a new mouse model of rhabdomyosarcoma that addresses an ongoing need for patient-relevant animal models to further translational research.

Augmenting chemotherapy with low-dose decitabine through an immune-independent mechanism.

The DNA methyltransferase inhibitor decitabine has classically been used to reactivate silenced genes and as a pretreatment for anticancer therapies. In a variation of this idea, this study explores the concept of adding low-dose decitabine (DAC) following administration of chemotherapy to bolster therapeutic efficacy. We find that addition of DAC following treatment with the chemotherapy agent gemcitabine improves survival and slows tumor growth in a mouse model of high-grade sarcoma. Unlike prior studies in epithelial tumor models, DAC did not induce a robust antitumor T cell response in sarcoma. Furthermore, DAC synergizes with gemcitabine independently of the immune system. Mechanistic analyses demonstrate that the combination therapy induces biphasic cell cycle arrest and apoptosis. Therapeutic efficacy was sequence dependent, with gemcitabine priming cells for treatment with DAC through inhibition of ribonucleotide reductase. This study identifies an apparently unique application of DAC to augment the cytotoxic effects of conventional chemotherapy in an immune-independent manner. The concepts explored in this study represent a promising paradigm for cancer treatment by augmenting chemotherapy through addition of DAC to increase tolerability and improve patient response. These findings have widespread implications for the treatment of sarcomas and other aggressive malignancies.

PRC2 loss drives MPNST metastasis and matrix remodeling.

The histone methyltransferase PRC2 plays a complex role in cancer. Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive sarcomas with frequent loss-of-function mutations in PRC2 that are associated with poor outcome. Here, we identify a critical role for PRC2 loss in driving MPNST metastasis. PRC2-dependent metastatic phenotypes included increased collagen-dependent invasion, upregulation of matrix-remodeling enzymes, and elevated lung metastasis in orthotopic mouse models. Furthermore, clinical sample analysis determined that PRC2 loss correlated with metastatic disease, increased fibrosis, and decreased survival in patients with MPNSTs. These results may have broad implications for PRC2 function across multiple cancers and provide a strong rationale for investigating potential therapies targeting ECM-remodeling enzymes and tumor fibrosis to improve outcomes in patients with MPNSTs.

Oncogenic RABL6A promotes NF1-associated MPNST progression in vivo.

Background: Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive sarcomas with complex molecular and genetic alterations. Powerful tumor suppressors CDKN2A and TP53 are commonly disrupted along with NF1, a gene that encodes a negative regulator of Ras. Many additional factors have been implicated in MPNST pathogenesis. A greater understanding of critical drivers of MPNSTs is needed to guide more informed targeted therapies for patients. RABL6A is a newly identified driver of MPNST cell survival and proliferation whose in vivo role in the disease is unknown. Methods: Using CRISPR-Cas9 targeting of Nf1 + Cdkn2a or Nf1 + Tp53 in the mouse sciatic nerve to form de novo MPNSTs, we investigated the biological significance of RABL6A in MPNST development. Terminal tumors were evaluated by western blot, qRT-PCR, and immunohistochemistry. Results: Mice lacking Rabl6 displayed slower tumor progression and extended survival relative to wildtype animals in both genetic contexts. YAP oncogenic activity was selectively downregulated in Rabl6-null, Nf1 + Cdkn2a lesions whereas loss of RABL6A caused upregulation of the CDK inhibitor, p27, in all tumors. Paradoxically, both models displayed elevated Myc protein and Ki67 staining in terminal tumors lacking RABL6A. In Nf1 + p53 tumors, cellular atypia and polyploidy were evident and increased by RABL6A loss. Conclusions: These findings demonstrate that RABL6A is required for optimal progression of NF1 mutant MPNSTs in vivo in both Cdkn2a and p53 inactivated settings. However, sustained RABL6A loss may provide selective pressure for unwanted alterations, including increased Myc, cellular atypia, and polyploidy, that ultimately promote a hyper-proliferative tumor phenotype akin to drug-resistant lesions.

Divergent immune landscapes of primary and syngeneic Kras-driven mouse tumor models.

Immune cells play critical functions in cancer, and mice with intact immune systems are vital to understanding tumor immunology. Both genetically engineered mouse models (GEMMs) and syngeneic cell transplant approaches use immunocompetent mice to define immune-dependent events in tumor development and progression. Due to their rapid and reproducible nature, there is expanded interest in developing new syngeneic tools from established primary tumor models. However, few studies have examined the extent that syngeneic tumors reflect the immune profile of their originating primary models. Here, we describe comprehensive immunophenotyping of two well-established GEMMs and four new syngeneic models derived from these parental primary tumors. To our knowledge, this is the first systematic analysis comparing immune landscapes between primary and orthotopic syngeneic tumors. These models all use the same well-defined human-relevant driver mutations, arise at identical orthotopic locations, and are generated in mice of the same background strain. This allows for a direct and focused comparison of tumor immune landscapes in carefully controlled mouse models. We identify key differences between the immune infiltrate of GEMM models and their corresponding syngeneic tumors. Most notable is the divergence of T cell populations, with different proportions of CD8+ T cells and regulatory T cells across several models. We also observe immune variation across syngeneic tumors derived from the same primary model. These findings highlight the importance of immune variance across mouse modeling approaches, which has strong implications for the design of rigorous and reproducible translational studies.

Assessing Changes and Associations in the Xanthomonas perforans Population Across Florida Commercial Tomato Fields Via a Statewide Survey.

Before 1991, Xanthomonas euvesicatoria was the causal agent of bacterial spot of tomato in Florida but was quickly replaced by X. perforans. The X. perforans population has changed in genotype and phenotype despite lack of a clear selection pressure. To determine the current Xanthomonas population in Florida, we collected 585 Xanthomonas strains from 70 tomato fields, representing 22 farms across eight counties, in the Florida tomato production region. Strains were isolated from 23 cultivars across eight seed producers and were associated with eight transplant facilities during the fall 2017 season. Our collection was phenotypically and genotypically characterized. Only X. perforans was identified, and all strains except one (99.8%) were tolerant to copper sulfate and 25% of strains were resistant to streptomycin sulfate. Most of the strains (99.3%) that were resistant to streptomycin sulfate were sequence type 1. The X. perforans population consisted of tomato races 3 (8%) and 4 (92%) and all three previously reported sequence types, ranging from 22 to 46% frequency. Approximately half of all strains, none of which were sequence type 2, produced bacteriocins against X. euvesicatoria. Effector profiles were highly variable among strains, which could impact the strains' host range. The effector xopJ4, which was previously thought to be conserved in X. perforans tomato pathogens, was absent in 19 strains. Nonmetric multidimensional scaling and network analyses show how strains and strain traits were associated with production system variables, including anonymized farms and transplant facilities. These analyses show that the composition of the Florida X. perforans population is diverse and complex.

Tumor Subtype Determines Therapeutic Response to Chimeric Polypeptide Nanoparticle-based Chemotherapy in Pten-deleted Mouse Models of Sarcoma.

PURPOSE: Nanoparticle-encapsulated drug formulations can improve responses to conventional chemotherapy by increasing drug retention within the tumor and by promoting a more effective antitumor immune response than free drug. New drug delivery modalities are needed in sarcomas because they are often chemoresistant cancers, but the rarity of sarcomas and the complexity of diverse subtypes makes it challenging to investigate novel drug formulations. EXPERIMENTAL DESIGN: New drug formulations can be tested in animal models of sarcomas where the therapeutic response of different formulations can be compared using mice with identical tumor-initiating mutations. Here, using Cre/loxP and CRISPR/Cas9 techniques, we generated two distinct mouse models of Pten-deleted soft-tissue sarcoma: malignant peripheral nerve sheath tumor (MPNST) and undifferentiated pleomorphic sarcoma (UPS). We used these models to test the efficacy of chimeric polypeptide doxorubicin (CP-Dox), a nanoscale micelle formulation, in comparison with free doxorubicin. RESULTS: The CP-Dox formulation was superior to free doxorubicin in MPNST models. However, in UPS tumors, CP-Dox did not improve survival in comparison with free doxorubicin. While CP-Dox treatment resulted in elevated intratumoral doxorubicin concentrations in MPNSTs, this increase was absent in UPS tumors. In addition, elevation of CD8+ T cells was observed exclusively in CP-Dox-treated MPNSTs, although these cells were not required for full efficacy of the CP nanoparticle-based chemotherapy. CONCLUSIONS: These results have important implications for treating sarcomas with nanoparticle-encapsulated chemotherapy by highlighting the tumor subtype-dependent nature of therapeutic response.

Distinct Tumor Microenvironments Are a Defining Feature of Strain-Specific CRISPR/Cas9-Induced MPNSTs.

The tumor microenvironment plays important roles in cancer biology, but genetic backgrounds of mouse models can complicate interpretation of tumor phenotypes. A deeper understanding of strain-dependent influences on the tumor microenvironment of genetically-identical tumors is critical to exploring genotype-phenotype relationships, but these interactions can be difficult to identify using traditional Cre/loxP approaches. Here, we use somatic CRISPR/Cas9 tumorigenesis approaches to determine the impact of mouse background on the biology of genetically-identical malignant peripheral nerve sheath tumors (MPNSTs) in four commonly-used inbred strains. To our knowledge, this is the first study to systematically evaluate the impact of host strain on CRISPR/Cas9-generated mouse models. Our data identify multiple strain-dependent phenotypes, including changes in tumor onset and the immune microenvironment. While BALB/c mice develop MPNSTs earlier than other strains, similar tumor onset is observed in C57BL/6, 129X1 and 129/SvJae mice. Indel pattern analysis demonstrates that indel frequency, type and size are similar across all genetic backgrounds. Gene expression and IHC analysis identify multiple strain-dependent differences in CD4+ T cell infiltration and myeloid cell populations, including M2 macrophages and mast cells. These data highlight important strain-specific phenotypes of genomically-matched MPNSTs that have implications for the design of future studies using similar in vivo gene editing approaches.

Development of a genome-informed loop-mediated isothermal amplification assay for rapid and specific detection of Xanthomonas euvesicatoria.

Bacterial spot (BS), caused by Xanthomonas euvesicatoria, X. vesicatoria, X. gardneri and X. perforans, is an economically important bacterial disease of tomato and pepper. Symptoms produced by all four species are nearly indistinguishable. At present, no point-of-care diagnostics exist for BS. In this research, we examined genomes of X. euvesicatoria, X. vesicatoria, X. gardneri, X. perforans and other species of Xanthomonas; the unique gene recG was chosen to design primers to develop a loop-mediated isothermal amplification (LAMP) assay to rapidly and accurately identify and differentiate X. euvesicatoria from other BS causing Xanthomonas sp. using a field-deployable portable BioRangerTM instrument. Specificity of the developed assay was tested against 39 strains of X. euvesicatoria and 41 strains of other species in inclusivity and exclusivity panels, respectively. The assay detection limit was 100 fg (~18 genome copies) of genomic DNA and 1,000 fg in samples spiked with tomato DNA. The assay unambiguously detected X. euvesicatoria in infected tomato plant samples. Concordant results were obtained when multiple operators performed the test independently. No false positives and false negatives were detected. The developed LAMP assay has numerous applications in diagnostics, biosecurity and disease management.

Snail1-dependent p53 repression regulates expansion and activity of tumour-initiating cells in breast cancer.

The zinc-finger transcription factor Snail1 is inappropriately expressed in breast cancer and associated with poor prognosis. While interrogating human databases, we uncovered marked decreases in relapse-free survival of breast cancer patients expressing high Snail1 levels in tandem with wild-type, but not mutant, p53. Using a Snail1 conditional knockout model of mouse breast cancer that maintains wild-type p53, we find that Snail1 plays an essential role in tumour progression by controlling the expansion and activity of tumour-initiating cells in preneoplastic glands and established tumours, whereas it is not required for normal mammary development. Growth and survival of preneoplastic as well as neoplastic mammary epithelial cells is dependent on the formation of a Snail1/HDAC1/p53 tri-molecular complex that deacetylates active p53, thereby promoting its proteasomal degradation. Our findings identify Snail1 as a molecular bypass that suppresses the anti-proliferative and pro-apoptotic effects exerted by wild-type p53 in breast cancer.

4D Tumorigenesis Model for Quantitating Coalescence, Directed Cell Motility and Chemotaxis, Identifying Unique Cell Behaviors, and Testing Anticancer Drugs.

A 4D high-resolution computer-assisted reconstruction and motion analysis system has been developed and applied to the long-term (14-30 days) analysis of cancer cells migrating and aggregating within a 3D matrix. 4D tumorigenesis models more closely approximate the tumor microenvironment than 2D substrates and, therefore, are improved tools for elucidating the interactions within the tumor microenvironment that promote growth and metastasis. The model we describe here can be used to analyze the growth of tumor cells, aggregate coalescence, directed cell motility and chemotaxis, matrix degradation, the effects of anticancer drugs, and the behavior of immune and endothelial cells mixed with cancer cells. The information given in this chapter is also intended to acquaint the reader with computer-assisted methods and algorithms that can be used for high-resolution 3D reconstruction and quantitative motion analysis.

Quantitative Motion Analysis in Two and Three Dimensions.

This chapter describes 2D quantitative methods for motion analysis as well as 3D motion analysis and reconstruction methods. Emphasis is placed on the analysis of dynamic cell shape changes that occur through extension and retraction of force generating structures such as pseudopodia and lamellipodia. Quantitative analysis of these structures is an underutilized tool in the field of cell migration. Our intent, therefore, is to present methods that we developed in an effort to elucidate mechanisms of basic cell motility, directed cell motion during chemotaxis, and metastasis. We hope to demonstrate how application of these methods can more clearly define alterations in motility that arise due to specific mutations or disease and hence, suggest mechanisms or pathways involved in normal cell crawling and treatment strategies in the case of disease. In addition, we present a 4D tumorigenesis model for high-resolution analysis of cancer cells from cell lines and human cancer tissue in a 3D matrix. Use of this model led to the discovery of the coalescence of cancer cell aggregates and unique cell behaviors not seen in normal cells or normal tissue. Graphic illustrations to visually display and quantify cell shape are presented along with algorithms and formulae for calculating select 2D and 3D motion analysis parameters.

A computer-assisted 3D model for analyzing the aggregation of tumorigenic cells reveals specialized behaviors and unique cell types that facilitate aggregate coalescence.

We have developed a 4D computer-assisted reconstruction and motion analysis system, J3D-DIAS 4.1, and applied it to the reconstruction and motion analysis of tumorigenic cells in a 3D matrix. The system is unique in that it is fast, high-resolution, acquires optical sections using DIC microscopy (hence there is no associated photoxicity), and is capable of long-term 4D reconstruction. Specifically, a z-series at 5 µm increments can be acquired in less than a minute on tissue samples embedded in a 1.5 mm thick 3D Matrigel matrix. Reconstruction can be repeated at intervals as short as every minute and continued for 30 days or longer. Images are converted to mathematical representations from which quantitative parameters can be derived. Application of this system to cancer cells from established lines and fresh tumor tissue has revealed unique behaviors and cell types not present in non-tumorigenic lines. We report here that cells from tumorigenic lines and tumors undergo rapid coalescence in 3D, mediated by specific cell types that we have named "facilitators" and "probes." A third cell type, the "dervish", is capable of rapid movement through the gel and does not adhere to it. These cell types have never before been described. Our data suggest that tumorigenesis in vitro is a developmental process involving coalescence facilitated by specialized cells that culminates in large hollow spheres with complex architecture. The unique effects of select monoclonal antibodies on these processes demonstrate the usefulness of the model for analyzing the mechanisms of anti-cancer drugs.

Mediated coalescence: a possible mechanism for tumor cellular heterogeneity.

Recently, we demonstrated that tumorigenic cell lines and fresh tumor cells seeded in a 3D Matrigel model, first grow as clonal islands (primary aggregates), then coalesce through the formation and contraction of cellular cables. Non-tumorigenic cell lines and cells from normal tissue form clonal islands, but do not form cables or coalesce. Here we show that as little as 5% tumorigenic cells will actively mediate coalescence between primary aggregates of majority non-tumorigenic or non-cancerous cells, by forming cellular cables between them. We suggest that this newly discovered, specialized characteristic of tumorigenic cells may explain, at least in part, why tumors contain primarily non-tumorigenic cells.

Huntingtin regulates Ca(2+) chemotaxis and K(+)-facilitated cAMP chemotaxis, in conjunction with the monovalent cation/H(+) exchanger Nhe1, in a model developmental system: insights into its possible role in Huntington׳s disease.

Huntington׳s disease is a neurodegenerative disorder, attributable to an expanded trinucleotide repeat in the coding region of the human HTT gene, which encodes the protein huntingtin. These mutations lead to huntingtin fragment inclusions in the striatum of the brain. However, the exact function of normal huntingtin and the defect causing the disease remain obscure. Because there are indications that huntingtin plays a role in Ca(2+) homeostasis, we studied the deletion mutant of the HTT ortholog in the model developmental system Dictyostelium discoideum, in which Ca(2+) plays a role in receptor-regulated behavior related to the aggregation process that leads to multicellular morphogenesis. The D. discoideum htt(-)-mutant failed to undergo both K(+)-facilitated chemotaxis in spatial gradients of the major chemoattractant cAMP, and chemotaxis up a spatial gradient of Ca(2+), but behaved normally in Ca(2+)-facilitated cAMP chemotaxis and Ca(2+)-dependent flow-directed motility. This was the same phenotypic profile of the null mutant of Nhel, a monovalent cation/H(+)exchanger. The htt(-)-mutant also failed to orient correctly during natural aggregation, as was the case for the Nhel mutant. Moreover, in a K(+)-based buffer the normal localization of actin was similarly defective in both htt(-) and nhe1(-) cells in a K(+)-based buffer, and the normal localization of Nhe1 was disrupted in the htt(-) mutant. These observations demonstrate that Htt and Nhel play roles in the same specific cation-facilitated behaviors and that Nhel localization is directly or indirectly regulated by Htt. Similar cation-dependent behaviors and a similar relationship between Htt and Nhe1 have not been reported for mammalian neurons and deserves investigation, especially as it may relate to Huntington׳s disease.

Myosin heavy chain kinases play essential roles in Ca2+, but not cAMP, chemotaxis and the natural aggregation of Dictyostelium discoideum.

Behavioral analyses of the deletion mutants of the four known myosin II heavy chain (Mhc) kinases of Dictyostelium discoideum revealed that all play a minor role in the efficiency of basic cell motility, but none play a role in chemotaxis in a spatial gradient of cAMP generated in vitro. However, the two kinases MhckA and MhckC were essential for chemotaxis in a spatial gradient of Ca(2+), shear-induced directed movement, and reorientation in the front of waves of cAMP during natural aggregation. The phenotypes of the mutants mhckA(-) and mhckC(-) were highly similar to that of the Ca(2+) channel/receptor mutant iplA(-) and the myosin II phosphorylation mutant 3XALA, which produces constitutively unphosphorylated myosin II. These results demonstrate that IplA, MhckA and MhckC play a selective role in chemotaxis in a spatial gradient of Ca(2+), but not cAMP, and suggest that Ca(2+) chemotaxis plays a role in the orientation of cells in the front of cAMP waves during natural aggregation.

The IplA Ca2+ channel of Dictyostelium discoideum is necessary for chemotaxis mediated through Ca2+, but not through cAMP, and has a fundamental role in natural aggregation.

During aggregation of Dictyostelium discoideum, nondissipating, symmetrical, outwardly moving waves of cAMP direct cells towards aggregation centers. It has been assumed that the spatial and temporal characteristics of the front and back of each cAMP wave regulate both chemokinesis and chemotaxis. However, during the period preceding aggregation, cells acquire not only the capacity to chemotax in a spatial gradient of cAMP, but also in a spatial gradient of Ca(2+). The null mutant of the putative IplA Ca(2+) channel gene, iplA(-), undergoes normal chemotaxis in spatial gradients of cAMP and normal chemokinetic responses to increasing temporal gradients of cAMP, both generated in vitro. However, iplA(-) cells lose the capacity to undergo chemotaxis in response to a spatial gradient of Ca(2+), suggesting that IplA is either the Ca(2+) chemotaxis receptor or an essential component of the Ca(2+) chemotaxis regulatory pathway. In response to natural chemotactic waves generated by wild-type cells, the chemokinetic response of iplA(-) cells to the temporal dynamics of the cAMP wave is intact, but the capacity to reorient in the direction of the aggregation center at the onset of each wave is lost. These results suggest that transient Ca(2+) gradients formed between cells at the onset of each natural cAMP wave augment reorientation towards the aggregation center. If this hypothesis proves correct, it will provide a more complex contextual framework for interpreting D. discoideum chemotaxis.

Ca2+ chemotaxis in Dictyostelium discoideum.

Using a newly developed microfluidic chamber, we have demonstrated in vitro that Ca(2+) functions as a chemoattractant of aggregation-competent Dictyostelium discoideum amoebae, that parallel spatial gradients of cAMP and Ca(2+) are more effective than either alone, and that cAMP functions as a stronger chemoattractant than Ca(2+). Effective Ca(2+) gradients are extremely steep compared with effective cAMP gradients. This presents a paradox because there is no indication to date that steep Ca(2+) gradients are generated in aggregation territories. However, given that Ca(2+) chemotaxis is co-acquired with cAMP chemotaxis during development, we speculate on the role that Ca(2+) chemotaxis might have and the possibility that steep, transient Ca(2+) gradients are generated during natural aggregation in the interstitial regions between cells.