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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.

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 movements of Dictyostelium discoideum amoebae translocating on a glass surface in the absence of chemoattractant have been reconstructed at 5-second intervals and motion analyzed by employing 3D-DIAS software. A morphometric analysis of pseudopods, the main cell body, and the uropod provides a comprehensive description of the basic motile behavior of a cell in four dimensions (4D), resulting in a list of 18 characteristics. A similar analysis of the myosin II phosphorylation mutant 3XASP reveals a role for the cortical localization of myosin II in the suppression of lateral pseudopods, formation of the uropod, cytoplasmic distribution of cytoplasm in the main cell body, and efficient motility. The results of the morphometric analysis suggest that pseudopods, the main cell body, and the uropod represent three motility compartments that are coordinated for efficient translocation. It provides a contextual framework for interpreting the effects of mutations, inhibitors, and chemoattractants on the basic motile behavior of D. discoideum. The generality of the characteristics of the basic motile behavior of D. discoideum must now be tested by similar 4D analyses of the motility of amoeboid cells of higher eukaryotic cells, in particular human polymorphonuclear leukocytes.

Extracellular Ca(++), a ubiquitous cation in the soluble environment of cells both free living and within the human body, regulates most aspects of amoeboid cell motility, including shape, uropod formation, pseudopod formation, velocity, and turning in Dictyostelium discoideum. Hence it affects the efficiency of both basic motile behavior and chemotaxis. Extracellular Ca(++) is optimal at 10 mM. A gradient of the chemoattractant cAMP generated in the absence of added Ca(++) only affects turning, but in combination with extracellular Ca(++), enhances the effects of extracellular Ca(++). Potassium, at 40 mM, can partially substitute for Ca(++). Mg(++), Mn(++), Zn(++), and Na(+) cannot. Extracellular Ca(++), or K(+), also induce the cortical localization of myosin II in a polar fashion. The effects of Ca(++), K(+) or a cAMP gradient do not appear to be similarly mediated by an increase in the general pool of free cytosolic Ca(++). These results suggest a model, in which each agent functioning through different signaling systems, converge to affect the cortical localization of myosin II, which in turn effects the behavioral changes leading to efficient cell motility and chemotaxis. Cell Motil. Cytoskeleton 2009. (c) 2009 Wiley-Liss, Inc.

2D- and 3D-Dynamic Image Analysis Systems (2D- and 3D-DIAS) for quantitative analysis of cell motility and chemotaxis are described. Particular attention is given to protocols that have proven useful in the quantitation of cell shape changes and pseudopod dynamics during basic cell motility (i.e. crawling in the absence of a chemotactic or other type of extracellular signal) and directed motion. In addition, methods provided, highlight the applicability of this approach to the accurate phenotypic characterizations of cytoskeletal mutations in Dictyostelium discoideum, cytoskeletal alterations in metastatic cells, and cytoskeletal defects in chemotactically defective polymorphonuclear neutrophils.

We have investigated the effects of inhibiting the expression of cofilin to understand its role in protrusion dynamics in metastatic tumor cells, in particular. We show that the suppression of cofilin expression in MTLn3 cells (an apolar randomly moving amoeboid metastatic tumor cell) caused them to extend protrusions from only one pole, elongate, and move rectilinearly. This remarkable transformation was correlated with slower extension of fewer, more stable lamellipodia leading to a reduced turning frequency. Hence, the loss of cofilin caused an amoeboid tumor cell to assume a mesenchymal-type mode of movement. These phenotypes were correlated with the loss of uniform chemotactic sensitivity of the cell surface to EGF stimulation, demonstrating that to chemotax efficiently, a cell must be able to respond to chemotactic stimulation at any region on its surface. The changes in cell shape, directional migration, and turning frequency were related to the re-localization of Arp2/3 complex to one pole of the cell upon suppression of cofilin expression.

It has been suggested that the phosphatydylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P(3)] phosphatase and tensin homolog PTEN plays a fundamental role in Dictyostelium discoideum chemotaxis. To identify that role, the behavior of a pten(-) mutant was quantitatively analyzed using two-dimensional and three-dimensional computer-assisted methods. pten(-) cells were capable of polarizing and translocating in the absence of attractant, and sensing and responding to spatial gradients, temporal gradients and natural waves of attractant. However, all of these responses were compromised (i.e. less efficient) because of the fundamental incapacity of pten(-) cells to suppress lateral pseudopod formation and turning. This defect was equally manifested in the absence, as well as presence, of attractant. PTEN, which is constitutively localized in the cortex of polarized cells, was found essential for the attractant-stimulated increase in cortical myosin II and F-actin that is responsible for the increased suppression of pseudopods during chemotaxis. PTEN, therefore, plays a fundamental role in the suppression of lateral pseudopod formation, a process essential for the efficiency of locomotion and chemotaxis, but not in directional sensing.

The chemotactic signal in Dictyostelium is a cAMP wave that is relayed over relatively large distances through a cell population during aggregation. Cells exhibit unique behaviors in response to the different spatial, temporal, and concentration components of the cAMP wave, suggesting that distinct signal transduction pathways are evoked in each of the various phases of the wave. For this reason, we designed a set of experimental protocols to test responses of normal and mutant Dictyostelium amoebae to the different components of a wave of chemoattractant. We then used computer-assisted two- (2D) and three-dimensional (3D) technologies (2D and 3D Dynamic Image Analysis System [DIAS]) for analysis of cells in the absence of a chemotactic signal (basic motile behavior) and in response to the temporal, spatial, and concentration components of the wave. As a result, we have elucidated parallel and independent pathways activated by specific phases of the cAMP wave. Likewise, human polymorphonuclear neutrophils (PMNs) respond to experimentally applied waves of the chemotactic peptide fMLP, and also exhibit discrete behavioral responses to the different phases. Using Dictyostelium as a paradigm, we applied our protocols to normal and diseased human PMNs and precisely defined a chemotactic defect. In this chapter, we describe methods for quantifying behaviors in Dictyostelium amoebae, PMNs, and other amoeboid cells using 2D and 3D DIAS. These methods are useful in the reconstruction and motion analysis of most migrating cells with transmitted and/or confocal microscopy.

Cyclic AMP (cAMP) functions as the extracellular chemoattractant in the aggregation phase of Dictyostelium development. There is some question, however, concerning what role, if any, it plays intracellularly in motility and chemotaxis. To test for such a role, the behavior of null mutants of acaA, the adenylyl cyclase gene that encodes the enzyme responsible for cAMP synthesis during aggregation, was analyzed in buffer and in response to experimentally generated spatial and temporal gradients of extracellular cAMP. acaA- cells were defective in suppressing lateral pseudopods in response to a spatial gradient of cAMP and to an increasing temporal gradient of cAMP. acaA- cells were incapable of chemotaxis in natural waves of cAMP generated by majority control cells in mixed cultures. These results indicate that intracellular cAMP and, hence, adenylyl cyclase play an intracellular role in the chemotactic response. The behavioral defects of acaA- cells were surprisingly similar to those of cells of null mutants of regA, which encodes the intracellular phosphodiesterase that hydrolyzes cAMP and, hence, functions opposite adenylyl cyclase A (ACA). This result is consistent with the hypothesis that ACA and RegA are components of a receptor-regulated intracellular circuit that controls protein kinase A activity. In this model, the suppression of lateral pseudopods in the front of a natural wave depends on a complete circuit. Hence, deletion of any component of the circuit (i.e., RegA or ACA) would result in the same chemotactic defect.

Sphingosine-1-phosphate (S-1-P) is a bioactive lipid that plays a role in diverse biological processes. It functions both as an extracellular ligand through a family of high-affinity G-protein-coupled receptors, and intracellularly as a second messenger. A growing body of evidence has implicated S-1-P in controlling cell movement and chemotaxis in cultured mammalian cells. Mutant D. discoideum cells, in which the gene encoding the S-1-P lyase had been specifically disrupted by homologous recombination, previously were shown to be defective in pseudopod formation, suggesting that a resulting defect might exist in motility and/or chemotaxis. To test this prediction, we analyzed the behavior of mutant cells in buffer, and in both spatial and temporal gradients of the chemoattractant cAMP, using computer-assisted 2-D and 3-D motion analysis systems. Under all conditions, S-1-P lyase null mutants were unable to suppress lateral pseudopod formation like wild-type control cells. This resulted in a reduction in velocity in buffer and spatial gradients of cAMP. Mutant cells exhibited positive chemotaxis in spatial gradients of cAMP, but did so with lowered efficiency, again because of their inability to suppress lateral pseudopod formation. Mutant cells responded normally to simulated temporal waves of cAMP but mimicked the temporal dynamics of natural chemotactic waves. The effect must be intracellular since no homologs of the S-1-P receptors have been identified in the Dictyostelium genome. The defects in the S-1-P lyase null mutants were similar to those seen in mutants lacking the genes for myosin IA, myosin IB, and clathrin, indicating that S-1-P signaling may play a role in modulating the activity or organization of these cytoskeletal elements in the regulation of lateral pseudopod formation., (2004 Wiley-Liss, Inc.)

To assess the role of myosin II heavy chain (MHC) phosphorylation in basic motility and natural chemotaxis, the Dictyostelium mhcA null mutant mhcA(-), mhcA(-) cells rescued with a myosin II gene that mimics the constitutively unphosphorylated state (3XALA) and mhcA(-) cells rescued with a myosin II gene that mimics the constitutively phosphorylated state (3XASP), were analyzed in buffer and in response to the individual spatial, temporal and concentration components of a cAMP wave using computer-assisted methods. Each mutant strain exhibited unique defects in cell motility and chemotaxis. Although mhcA(-) cells could crawl with some polarity and showed chemotaxis with highly reduced efficiency in a spatial gradient of cAMP, they were very slow, far less polar and more three-dimensional than control cells. They were also incapable of responding to temporal gradients of cAMP, of chemotaxis in a natural wave of cAMP or streaming late in aggregation. 3XASP cells were faster and chemotactically more efficient than mhcA(-) cells, but still incapable of responding to temporal gradients of cAMP, chemotaxis in natural waves of cAMP or streaming late in aggregation. 3XALA cells were fast, were able to respond to temporal gradients of cAMP, and responded to natural waves of cAMP. However, they exhibited a 50% reduction in chemotactic efficiency, could not stream late in aggregation and could not enter the streams of control cells in mixed cultures. F-actin staining further revealed that while the presence of unphosphorylated MHC was essential for the increase in F-actin in the cytoplasm in response to the increasing temporal gradient of cAMP in the front of a natural wave, the actual dephosphorylation event was essential for the associated increase in cortical F-actin. The results of these studies indicate that MHC phosphorylation-dephosphorylation, like myosin II regulatory light chain phosphorylation-dephosphorylation, represents a potential downstream target of the regulatory cascades emanating from the different phases of the wave.

To define the role that RasC plays in motility and chemotaxis, the behavior of a rasC null mutant, rasC-, in buffer and in response to the individual spatial, temporal, and concentration components of a natural cyclic AMP (cAMP) wave was analyzed by using computer-assisted two-dimensional and three-dimensional motion analysis systems. These quantitative studies revealed that rasC- cells translocate at the same velocity and exhibit chemotaxis up spatial gradients of cAMP with the same efficiency as control cells. However, rasC- cells exhibit defects in maintaining anterior-posterior polarity along the substratum and a single anterior pseudopod when translocating in buffer in the absence of an attractant. rasC- cells also exhibit defects in their responses to both the increasing and decreasing temporal gradients of cAMP in the front and the back of a wave. These defects result in the inability of rasC- cells to exhibit chemotaxis in a natural wave of cAMP. The inability to respond normally to temporal gradients of cAMP results in defects in the organization of the cytoskeleton, most notably in the failure of both F actin and myosin II to exit the cortex in response to the decreasing temporal gradient of cAMP in the back of the wave. While the behavioral defect in the front of the wave is similar to that of the myoA-/myoF- myosin I double mutant, the behavioral and cytoskeletal defects in the back of the wave are similar to those of the S13A myosin II regulatory light-chain phosphorylation mutant. Expression array data support the premise that the behavioral defects exhibited by the rasC- mutant are the immediate result of the absence of RasC function., (Copyright 2004 American Society for Microbiology)

The myosin regulatory light chain (RLC) of Dictyostelium discoideum is phosphorylated at a single serine site in response to chemoattractant. To investigate the role of the phosphorylation of RLC in both motility and chemotaxis, mutants were generated in which the single phosphorylatable serine was replaced with a nonphosphorylatable alanine. Several independent clones expressing the mutant RLC in the RLC null mutant, mlcR(-), were obtained. These S13A mutants were subjected to high resolution computer-assisted motion analysis to assess the basic motile behavior of cells in the absence of a chemotatic signal, and the chemotactic responsiveness of cells to the spatial, temporal and concentration components of natural cAMP waves. In the absence of a cAMP signal, mutant cells formed lateral pseudopods less frequently and crawled faster than wild-type cells. In a spatial gradient of cAMP, mutant cells chemotaxed more efficiently than wild-type cells. In the front of simulated temporal and natural waves of cAMP, mutant cells responded normally by suppressing lateral pseudopod formation. However, unlike wild-type cells, mutant cells did not lose cellular polarity at the peak and in the back of either wave. Since depolarization at the peak and in the descending phase of the natural wave is necessary for efficient chemotaxis, this deficiency resulted in a decrease in the capacity of S13A mutant cells to track natural cAMP waves relayed by wild-type cells, and in the fragmentation of streams late in mutant cell aggregation. These results reveal a regulatory pathway induced by the peak and back of the chemotactic wave that alters RLC phosphorylation and leads to cellular depolarization. We suggest that depolarization requires myosin II rearrangement in the cortex facilitated by RLC phosphorylation, which increases myosin motor function.

In the natural aggregation process, Dictyostelium amoebae relay the cAMP signal outwardly through the cell population as symmetric, nondissipating waves. Each cell in turn responds in a specific manner to the different phases of the wave. In the front of each wave, each cell experiences an increasing temporal gradient and positive spatial gradient of cAMP; at the peak of each wave, each cell experiences a cAMP concentration inhibitory to locomotion; and in the back of each wave, each cell experiences a decreasing temporal and negative spatial gradient of cAMP. Protocols are described to analyze the basic motile behavior of mutant cells in the absence of a chemotactic signal, and to test the responsiveness of mutant cells to the individual temporal, spatial and concentration components of a natural wave. The results of such an analysis can then be used to develop realistic models of cell motility and chemotaxis. Examples are described in which this contextual framework has been applied to mutant cell lines. The results of these mutant studies result in a model in which independent parallel regulatory pathways emanating from different phases of the wave effect different phase-specific behaviors.

Cell behavior is three-dimensional (3-D), even when it takes place on a flat surface. Migrating cells form pseudopods on and off the substratum, and the cell body undergoes height changes associated with a 1 min behavior cycle. Inside the cell, the nucleus has a 3-D migratory cycle, and vesicles move up and down in the z-axis as a cell locomotes. For these reasons, the two-dimensional (2-D) analysis of cellular and subcellular behavior is, in many cases, inadequate. We have, therefore, developed 3-D motion analysis systems that reconstruct the cell surface, nucleus, pseudopods, and vesicles of living, crawling cells in 3-D at time intervals as short as 1 s, and compute more than 100 parameters of motility and dynamics morphology at 1-s intervals. We are now in the process of developing a multimode reconstruction system that will allow us to reconstruct and analyze fluorescently tagged molecular complexes within the differential interference contrast-imaged subcellular architecture of a crawling cell. These evolving technologies should find wide application for a host of biomedical problems.

Forced expression of the chimeric human fibroblast tropomyosin 5/3 (hTM5/3) in CHO cell was previously shown to affect cytokinesis [Warren et al., 1995: J. Cell Biol. 129:697-708]. To further investigate the phenotypic consequences of misexpression, we have compared mitotic spindle organization and dynamic 2D and 3D shape changes during mitosis in normal cells and in a hTM5/3 misexpressing (mutant) cell line. Immunofluorescence microscopy of wild type and mutant cells stained with monoclonal anti-tubulin antibody revealed that the overall structures of mitotic spindles were not significantly different. However, the axis of the mitotic spindle in mutant cells was more frequently misaligned with the long axis of the cell than that of wild type cells. To assess behavioral differences during mitosis, wild type and mutant cells were reconstructed in 2D and 3D and motion analyzed with the computer-assisted 2D and 3D Dynamic Image Analysis Systems (2D-DIAS, 3D-DIAS). Mutant cells abnormally formed large numbers of blebs during the later stages of mitosis and took longer to proceed from the start of anaphase to the start of cytokinesis. Furthermore, each mutant cell undergoing mitosis exhibited greater shape complexity than wild type cells, and in every case lifted one of the two evolving daughter cells off the substratum and abnormally twisted. These results demonstrate that misexpression of hTM5/3 in CHO cells leads to morphological instability during mitosis. Misexpression of hTM5/3 interferes with normal tropomyosin function, suggesting in turn that tropomyosin plays a role through its interaction with actin microfilaments in the regulation of the contractile ring, in the localized suppression of blebbing, in the maintenance of polarity and spatial symmetry during cytokinesis, and in cell spreading after cytokinesis is complete., (Copyright 2000 Wiley-Liss, Inc.)

Although the traditional role of clathrin has been in vesicle trafficking and the internalization of receptors, a novel role in cytokinesis was recently revealed in an analysis of a clathrin-minus Dictyostelium mutant (chc(-)). chc(-) cells grown in suspension were demonstrated to be defective in assembling myosin II into a normal contractile ring. To test whether this defect reflected a more general one of cytoskeletal dysfunction, chc(-) cells were analyzed for cell polarity, pseudopod formation, uropod stability, cell locomotion, chemotaxis, cytoskeletal organization and vesicle movement. chc(-) cells crawled, chemotaxed, localized F-actin in pseudopods, organized their microtubule cytoskeleton in a relatively normal fashion and exhibited normal vesicle dynamics. Although chc(-) cells extended pseudopods from the anterior half of the cell with the same frequency as normal chc(+) cells, they extended pseudopods at twice the normal frequency from the posterior half of the cell. The uropods of chc(-) cells also exhibited spatial instability. These defects resulted in an increase in roundness, a reduction in polarity, a reduction in velocity, a dramatic increase in turning, a high frequency of 180 degrees direction reversals and a decrease in the efficiency of chemotaxis. All defects were reversed in a rescued strain. These results are the first to suggest a novel role for clathrin in cell polarity, pseudopod formation, uropod stability and locomotion. It is hypothesized that clathrin functions to suppress pseudopod formation and to stabilize the uropod in the posterior half of a crawling cell, two behavioral characteristics that are essential for the maintenance of cellular polarity, efficient locomotion and efficient chemotaxis.

The actin binding protein ABP-120 has been proposed to cross-link actin filaments in nascent pseudopods, in a step required for normal pseudopod extension in motile Dictyostelium amoebae. To test this hypothesis, cell lines that lack ABP-120 were created independently either by chemical mutagenesis or homologous recombination. Different phenotypes were reported in these two studies. The chemical mutant shows only a subtle defect in actin cross-linking, while the homologous recombinant mutants show profound defects in actin cross-linking, cytoskeletal structure, pseudopod number and size, cell motility and chemotaxis and, as shown here, phagocytosis. To resolve the controversy as to what the ABP-120- phenotype is, ABP-120 was re-expressed in an ABP-120- cell line created by homologous recombination. Two independently "rescued" cell lines that express wild-type levels of ABP-120 were analyzed. In both rescued cell lines, actin incorporation into the cytoskeleton, pseudopod formation, cell morphology, instantaneous velocity, phagocytosis, and chemotaxis were restored to wild-type levels. There is no alteration in the expression levels of several related actin binding proteins in either the original ABP-120- cell line or in the rescued cell lines, leading to the conclusion that neither the aberrant phenotype observed in ABP-120- cells nor the normal phenotype reasserted in rescued cells can be attributed to alterations in the levels of other abundant and related actin binding proteins. Re-expression of ABP-120 in ABP-120- cells reestablishes normal structural and behavioral parameters, demonstrating that the severity and properties of the structural and behavioral defects of ABP-120- cell lines produced by homologous recombination are the direct result of the absence of ABP-120.

The streamer F (StmF) mutant of Dictyostelium discoideum is defective in cGMP-phosphodiesterase activity. In early aggregation territories, when individual cells chemotax toward aggregation centers prior to streaming, the average periodicity of surging of StmF cells is half that of wild-type cells. In addition, in the period between surges, which has been interpreted to include the peak and back of the wave, StmF cells abnormally remain nonmotile and retain their elongate shape. In contrast, in the period between surges wild-type cells form pseudopods randomly around their cell perimeter and take on an amorphous shape. Using a newly developed protocol for vitally staining and tracking individual mutant cells in unstained wild-type aggregation territories and individual wild-type cells in unstained StmF aggregation territories, we have found that mutant cells behave normally (i.e., like their predominant wild-type neighbors) in the deduced back of waves generated by wild-type cells. Conversely, wild-type cells behave aberrantly (i.e., like their predominant StmF neighbors) in the deduced back of waves generated by mutant cells. These results suggest that the defective behavior of StmF cells in early StmF aggregation territories is not due to a single cell defect in responsiveness, but, rather, is due either to the genesis of an aberrant cAMP wave or to the accumulation of a molecule which interferes with normal behavior in the back of the wave.

The myoA gene of Dictyostelium is a member of a gene family of unconventional myosins. The myosin Is share homologous head and basic domains, but the myoA gene product lacks the glycine-, proline-, alanine-rich and src homology 3 domains typical of several of the other myosin Is. A mutant strain of Dictyostelium lacking a functional myoA gene was produced by gene targeting, and the motility of this strain in buffer and a spatial gradient of the chemoattractant cyclic AMP was analyzed by computer-assisted methods. The myoA- cells have a normal elongate morphology in buffer but exhibit a decrease in the instantaneous velocity of cellular translocation, an increase in the frequency of lateral pseudopod formation, and an increase in turning. In a spatial gradient, in which the frequency of pseudopod formation is depressed, myoA- cells exhibit positive chemotaxis but still turn several times more frequently than control cells. These results demonstrate that the other members of the unconventional myosin family do not fully compensate for the loss of functional myoA gene product. Surprisingly, the phenotype of the myoA- strain closely resembles that of the myoB- strain, suggesting that both play a role in the frequency of pseudopod formation and turning during cellular translocation.

The actin-binding protein ABP-120 has been proposed to play a role in cross-linking F-actin filaments during pseudopod formation in motile Dictyostelium amebas. We have tested this hypothesis by analyzing the phenotype of mutant cell lines which do not produce ABP-120. Two different transformation vectors capable of targeted disruption of the ABP-120 gene locus have been constructed using a portion of an ABP-120 cDNA clone. Three independent cell lines with different disruption events have been obtained after transformation of amebas with these vectors. The disruption of the ABP-120 gene by vector sequences results in either the production of a small amount of truncated ABP-120 or no detectable protein at all. The phenotypes of two different clones lacking ABP-120, generated in strains AX3 and AX4, have been characterized and show identical results. ABP-120- cells tend to remain rounder before and after cAMP stimulation, and do not reextend pseudopods normally after rapid addition of cAMP. In addition, ABP-120- cells translocating in buffer exhibit defects in both the rate and extent of pseudopod formation. The amount of F-actin cross-linked into the cytoskeleton after cAMP stimulation of ABP-120- cells is reduced at times when ABP-120 has been shown to be incorporated into the cytoskeleton, and this correlates temporally with the absence of reextension of pseudopods after cAMP stimulation. The instantaneous velocity is significantly reduced both before and after cAMP stimulation in the ABP-120- cells, and the cells show decreased chemotactic efficiency compared to ABP-120+ controls. This phenotype is consistent with a role for ABP-120 in pseudopod extension by cross-linking actin filaments as proposed by the "cortical expansion model" (Condeelis, J., A. Bresnick, M. Demma, C. Dharmawardhane, R. Eddy, A. L. Hall, R. Sauterer, and V. Warren. 1990. Dev. Genet. 11:333-340).

Dictyostelium amebae have been engineered by homologous recombination of a truncated copy of the myosin heavy chain gene (heavy meromyosin (HMM) cells) and by transformation with a vector encoding an antisense RNA to myosin heavy chain mRNA (mhcA cells) so that they lack native myosin heavy chain protein. In the former case, cells synthesize only the heavy meromyosin portion of the protein and in the latter case they synthesize negligible amounts of the protein. Surprisingly, it was demonstrated that both cell lines are viable and motile. In order to compare the motility of these cells with normal cells, the newly developed computer-assisted Dynamic Morphology System (DMS) was employed. The results demonstrate that the average HMM or mhcA ameba moves at a rate of translocation less than half that of normal cells. It is rounder and less polar than a normal cell, and exhibits a rate of cytoplasmic expansion and contraction roughly half that of normal cells. In a spatial gradient of cAMP, the average ameba of HMM or mhcA exhibits a chemotactic index of +0.10 or less, compared to the chemotactic index of +0.50 exhibited by normal cells. Finally, the initial area, rate of expansion, and final area of pseudopods are roughly half that of normal cells. The five fastest HMM amebae (out of 35 analyzed in detail) moved at an average rate of translocation equal to that of normal amebae, and exhibited an average chemotactic index of +0.34. In addition, the average rate of cytoplasmic flow in fast HMM cells was equal to that of the average normal ameba. However, fast HMM amebae still exhibited the same defects in pseudopod formation that were exhibited by the entire HMM cell population. These results suggest that myosin heavy chain is involved in the "fine tuning" and efficiency of pseudopod formation, but is not essential for the basic behavior of pseudopod expansion.