Plant lectin can target receptors containing sialic Acid, exemplified by podoplanin, to inhibit transformed cell growth and migration

Cancer is a leading cause of death of men and women worldwide. Tumor cell motility contributes to metastatic invasion that causes the vast majority of cancer deaths. Extracellular receptors modified by α2,3-sialic acids that promote this motility can serve as ideal chemotherapeutic targets. For example, the extracellular domain of the mucin receptor podoplanin (PDPN) is highly O-glycosylated with α2,3-sialic acid linked to galactose. PDPN is activated by endogenous ligands to induce tumor cell motility and metastasis. Dietary lectins that target proteins containing α2,3-sialic acid inhibit tumor cell growth. However, anti-cancer lectins that have been examined thus far target receptors that have not been identified. We report here that a lectin from the seeds of Maackia amurensis (MASL) with affinity for O-linked carbohydrate chains containing sialic acid targets PDPN to inhibit transformed cell growth and motility at nanomolar concentrations. Interestingly, the biological activity of this lectin survives gastrointestinal proteolysis and enters the cardiovascular system to inhibit melanoma cell growth, migration, and tumorigenesis. These studies demonstrate how lectins may be used to help develop dietary agents that target specific receptors to combat malignant cell growth.     

Autonomous right-screw rotation of growth cone filopodia drives neurite turning

The direction of neurite elongation is controlled by various environmental cues. However, it has been reported that even in the absence of any extrinsic directional signals, neurites turn clockwise on two-dimensional substrates. In this study, we have discovered autonomous rotational motility of the growth cone, which provides a cellular basis for inherent neurite turning. We have developed a technique for monitoring three-dimensional motility of growth cone filopodia and demonstrate that an individual filopodium rotates on its own longitudinal axis in the right-screw direction from the viewpoint of the growth cone body. We also show that the filopodial rotation involves myosins Va and Vb and may be driven by their spiral interactions with filamentous actin. Furthermore, we provide evidence that the unidirectional rotation of filopodia causes deflected neurite elongation, most likely via asymmetric positioning of the filopodia onto the substrate. Although the growth cone itself has been regarded as functionally symmetric, our study reveals the asymmetric nature of growth cone motility.     

Traveling bands of chemotactic bacteria in the context of population growth

A mathematical model for traveling bands of motile and chemotactic bacteria in the presence of cell growth and death is examined. It is found that asymptotic traveling wave solutions exist in the absence of chemotaxis, due to the balance of growth, death and random motility. Thus random motility confers the ecological advantage of population propagation through migration into nutrient-rich regions. The presence of chemotaxis amplifies this advantage by moving more cells into higher nutrient concentration regions, resulting in larger and faster bands. Therefore there seem to be two types of traveling bands that can be attained by chemotactic bacteria in the presence of growth and death: (1) these growth/death/motility bands; and (2) pure chemotactic ‘Keller-Segel'-type bands. Comparison to experimental observations by Chapman in 1973 indicate that the latter seem to be formed. The relationship between these two types of solution is at present uncertain. The growth/death/motility bands may have relevance on longer time or distance scales characteristic of microbial ecological systems.     

Substrate-cytoskeletal coupling as a mechanism for the regulation of growth cone motility and guidance

Growth cones are highly motile structures at the end of neuronal processes, capable of receiving multiple types of guidance cues and transducing them into directed axonal growth. Thus, to guide the axon toward the appropriate target cell, the growth cone carries out different functions: it acts as a sensor, signal transducer, and motility device. An increasing number of molecular components that mediate axon guidance have been characterized over the past years. The vast majority of these molecules include proteins that act as guidance cues and their respective receptors. In addition, more and more signaling and cytoskeleton-associated proteins have been localized to the growth cone. Furthermore, it has become evident that growth cone motility and guidance depends on a dynamic cytoskeleton that is regulated by incoming guidance information. Current and future research in the growth cone field will be focussed on how different guidance cues transmit their signals to the cytoskeleton and change its dynamic properties to affect the rate and direction of growth cone movement. In this review, we discuss recent evidence that cell adhesion molecules can regulate growth cone motility and guidance by a mechanism of substrate-cytoskeletal coupling.     

Effect of potent and selective inhibitors of the Grb2 SH2 domain on cell motility.

Cell motility has been correlated both with oncogenic invasiveness and metastatic potential. The development of selective inhibitors of motility has thus great potential importance. Grb2 is a SH2/SH3 domain-containing adaptor protein that links growth factor receptor tyrosine kinases to the Ras signaling pathway. We have developed specific small molecule inhibitors of the Grb2 SH2 domain as potential leads for drug discovery. Synthesis of the inhibitors and their effects on growth factor-induced growth in cells have been reported previously. In the current study, we establish that these inhibitors inhibit hepatocyte growth factor/scatter factor-induced A431 and Madin-Darby canine kidney cell motility and various cell motility-related events, including epidermal growth factor-induced ruffling of A431 cells and epidermal growth factor-induced translocation of the small GTPase Rac in these cells. We demonstrate for the first time a direct role for Grb2 in cell motility and indicate a new avenue for cancer therapeutics.     

Insulin-like growth factor II acts as an autocrine growth and motility factor in human rhabdomyosarcoma tumors

Rhabdomyosarcoma is the most common soft tissue sarcoma of childhood and appears to arise from developing striated muscle-forming cells. Since insulin-like growth factor II (IGF-II) is involved in normal muscle growth and maturation and elevated IGF-II mRNA levels have previously been reported in rhabdomyosarcomas, we have been studying the possible role of IGF-II in the unregulated growth and invasive potential of these embryonal tumors. In this study, we demonstrate that 13 of 14 rhabdomyosarcoma tumors express high levels of IGF-II mRNA relative to normal adult muscle and also express mRNA for the type I IGF receptors on their cell surface, the receptor thought to mediate the effects of IGF-II on muscle cells. We have established several rhabdomyosarcoma cell lines in mitogen-free media and demonstrate that these cells express type I IGF receptors on their cell surface and secrete IGF-II into the media. Exogenous IGF-II is able to stimulate cellular motility in these cell lines as assayed in a modified Boyden chamber. Finally, alpha IR-3, a type I receptor antagonist, inhibits the growth of these cell lines in serum-free media but does not inhibit IGF-II-induced motility of these cells. These data suggest that endogenously produced IGF-II functions as an autocrine growth and motility factor in many rhabdomyosarcoma tumors. The mitogenic actions of IGF-II are mediated through a domain of the type I IGF receptor that is blocked by alpha IR-3. IGF-II-induced motility may be mediated through an alternative signaling pathway.     

Hepatocyte growth factor induces epithelial cell motility through transactivation of the epidermal growth factor receptor

Hepatocyte growth factor (HGF) is a potent inducer of motility in epithelial cells. Since we have previously found that activation of the epidermal growth factor receptor (EGFR) is an absolute prerequisite for induction of motility of corneal epithelial cells after wounding, we investigated whether induction of motility in response to HGF is also dependent on activation of the EGFR. We now report that HGF induces transactivation of the EGFR in an immortalized line of corneal epithelial cells, in human skin keratinocytes, and in Madin-Darby canine kidney cells. EGFR activation is unconditionally required for induction of motility in corneal epithelial cells, and for induction of a fully motile phenotype in Madin-Darby canine kidney cells. Activation of the EGFR occurs through amphiregulin and heparin-binding epidermal growth factor-like growth factor. Early after HGF stimulation, blocking EGFR activation does not inhibit extracellular-signal regulated kinase 1/2 (ERK1/2) activation by HGF, but the converse is seen after approximately 1 h, indicating the existence of EGFR-dependent and -independent routes of ERK1/2 activation. In summary, HGF induces transactivation of the EGFR in epithelial cells, and this is a prerequisite for induction of full motility.     

Correlating single cell motility with population growth dynamics for flagellated bacteria

Many bacteria used for biotechnological applications are naturally motile. Their "bio-nanopropeller" driven movement allows searching for better environments in a process called chemotaxis. Since bacteria are extremely small in size compared to the bulk fluid volumes in bioreactors, single cell motility is not considered to influence bioreactor operations. However, with increasing interest in localized fluid flow inside reactors, it is important to ask whether individual motility characteristics of bacteria are important in bioreactor operations. The first step in this direction is to try to correlate single cell measurements with population data of motile bacteria in a bioreactor. Thus, we observed the motility behavior of individual bacterial cells, using video microscopy with 33 ms time resolution, as a function of population growth dynamics of batch cultures in shake flasks. While observing the motility behavior of the most intensively studied bacteria, Escherichia coli, we find that overall bacterial motility decreases with progression of the growth curve. Remarkably, this is due to a decrease in a specific motility behavior called "running". Our results not only have direct implications on biofilm formations, but also provide a new direction in bioprocess design research highlighting the role of individual bacterial cell motility as an important parameter.     

Membrane voltage and neurotransmitter regulation of neuronal growth cone motility

The neurotransmitters serotonin and dopamine inhibit growth cone motility and neurite elongation of specific identified neurons of the pond snail Helisoma. Similarly, experimentally evoked action potentials inhibit motility of these growth cones. Here we explore the possibility that the motility- and elongation-inhibiting actions of serotonin and dopamine derive from the electrophysiological responses of the respective neurons. Evidence of three types in support of this hypothesis is presented: (1) Only those identified neurons for which motility is inhibited by serotonin or dopamine respond to the transmitter with sustained electrical excitation. (2) The magnitude of the electrical excitation response correlates with the degree of inhibition of growth cone motility. (3) The injection of hyperpolarizing current enables motility to continue as in the absence of transmitters. We conclude that membrane voltage is an important level of control of growth cone motility, at which neurotransmitters exert a regulatory influence.     

Effects of cell motility and chemotaxis on microbial population growth.

A mathematical model is developed to elucidate the effects of biophysical transport processes (nutrient diffusion, cell motility, and chemotaxis) along with biochemical reaction processes (cell growth and death, nutrient uptake) upon steady-state bacterial population growth in a finite one-dimensional region. The particular situation considered is that of growth limitation by a nutrient diffusing from an adjacent phase not accessible to the bacteria. It is demonstrated that the cell motility and chemotaxis properties can have great influence on steady-state population size. In fact, motility effects can be as significant as growth kinetic effects, in a manner analogous to diffusion- and reaction-limited regimes in chemically reacting systems. In particular, the following conclusions can be drawn from our analysis for bacterial populations growing at steady-state in a confined, unmixed region: (a) Random motility may lead to decreased population density; (b) chemotaxis can allow increased population density if the chemotactic response is large enough; (c) a species with superior motility properties can outgrow a species with superior growth kinetic properties; (d) motility effects become greater as the size of the confined growth region increases; and (e) motility effects are diminished by significant mass-transfer limitation of the nutrient from the adjacent source phase. The relationships of these results for populations to previous conclusions for individual cells is discussed, and implications for microbial competition are suggested.     

Using experimental evolution to explore natural patterns between bacterial motility and resistance to bacteriophages

Resistance of bacteria to phages may be gained by alteration of surface proteins to which phages bind, a mechanism that is likely to be costly as these molecules typically have critical functions such as movement or nutrient uptake. To address this potential trade-off, we combine a systematic study of natural bacteria and phage populations with an experimental evolution approach. We compare motility, growth rate and susceptibility to local phages for 80 bacteria isolated from horse chestnut leaves and, contrary to expectation, find no negative association between resistance to phages and bacterial motility or growth rate. However, because correlational patterns (and their absence) are open to numerous interpretations, we test for any causal association between resistance to phages and bacterial motility using experimental evolution of a subset of bacteria in both the presence and absence of naturally associated phages. Again, we find no clear link between the acquisition of resistance and bacterial motility, suggesting that for these natural bacterial populations, phage-mediated selection is unlikely to shape bacterial motility, a key fitness trait for many bacteria in the phyllosphere. The agreement between the observed natural pattern and the experimental evolution results presented here demonstrates the power of this combined approach for testing evolutionary trade-offs.     

Expression of unconventional myosin genes during neuronal development in zebrafish

Neuronal migration and growth cone motility are essential aspects of the development and maturation of the nervous system. These cellular events result from dynamic changes in the organization and function of the cytoskeleton, in part due to the activity of cytoskeletal motor proteins such as myosins. Although specific myosins such as Myo2 (conventional or muscle myosin), Myo1, and Myo5 have been well characterized for roles in cell motility, the roles of the majority of unconventional (other than Myo2) myosins in cell motility events have not been investigated. To address this issue, we have undertaken an analysis of unconventional myosins in zebrafish, a premier model for studying cellular and growth cone motility in the vertebrate nervous system. We describe the characterization and expression patterns of several members of the unconventional myosin gene family. Based on available genomic sequence data, we identified 18 unconventional myosin- and 4 Myo2-related genes in the zebrafish genome in addition to previously characterized myosin (1, 2, 3, 5, 6, 7) genes. Phylogenetic analyses indicate that these genes can be grouped into existing classifications for unconventional myosins from mouse and man. In situ hybridization analyses using EST probes for 18 of the 22 identified genes indicate that 11/18 genes are expressed in a restricted fashion in the zebrafish embryo. Specific myosins are expressed in particular neuronal or neuroepithelial cell types in the developing zebrafish nervous system, spanning the periods of neuronal differentiation and migration, and of growth cone guidance and motility.     

Differential modulation of hepatocyte growth factor-stimulated motility by transforming growth factor β1 on rat liver epithelial cells in vitro

We have previously shown that transforming growth factor-β1 (TGF-β1) enhances the epidermal growth factor- (EGF) and transforming growth factor-α (TGF-α)-stimulated motility of rat hepatocytes in an extracellular matrix (ECM)-dependent fashion (Stolz and Michalopoulos, 1997, J. Cell. Physiol., 170:57–68). We have extended this study to examine the effects of TGF-β1 on hepatocyte growth factor (HGF) and EGF-stimulated motility of rat nonparenchymal liver epithelial cells (RLECs) in vitro and determined that chemotaxis, scattering, and monolayer wound healing by EGF was synergistically enhanced by TGF-β1 on all ECMs examined. However, HGF-based motility, unlike EGF-stimulated motility, was modulated in an assay-dependent manner by TGF-β1. HGF-stimulated chemotaxis was dramatically decreased by addition of TGF-β1, but wound healing was synergistically enhanced by TGF-β1 on all ECMs examined. HGF-based scattering was not consistently affected by TGF-β1 on any ECM tested except on laminin, where scattering was often reduced by the concomitant addition of TGF-β1. TGF-β1 enhanced the motility associated with monolayer wound healing by HGF or EGF independent of DNA synthesis, because tritiated thymidine uptake was consistently reduced by 60% in the presence of TGF-β1. The data indicate that HGF and EGF motility do not follow redundant signal-transduction pathways and that specific growth factor motility-related events, as measured by wound healing, scattering, and chemotaxis, are modulated independently by ECM and TGF-β1. J. Cell. Physiol. 175:30–40, 1998. © 1998 Wiley-Liss, Inc.     

Semen characteristics of genetically identical quadruplet bulls

Modern cloning methods have become an important technology in artificial insemination which is used to create and maintain pools of genetically superior bull semen. In this study, semen from four identical quadruplet bulls (Q(1), Q(2), Q(3), and Q(4)) produced by blastomere separation was analyzed to evaluate the differences in reproductive potential, if any, that existed between the identical quadruplet siblings. Analysis of fresh semen collected from 1994 to 1996, showed lower progressive motility and lower sperm concentration for one bull (Q(3)) compared to his identical brothers (P<0.05). Semen characteristics following freezing-thawing procedures have also been tested for these quadruplet bulls. The percentage of motility, progressive motility, and mean path velocity were lower in Q(4) compared with Q(1). Moreover, intracellular calcium level and P25b level (P25b is a sperm surface protein proposed to be a potential bull fertility marker) were lower in Q(4) compared with his siblings (P<0.05). Cryodamage to Q(4)'s frozen-thawed spermatozoa were confirmed by a lower percentage of embryo development after in vitro fertilization. Thus, the higher instability of cryopreserved spermatozoa from Q(4) and the lower semen production of Q(3), compared to their siblings, indicate that differences in semen characteristics can indeed exist among genetically identical animals produced by blastomere separation.     

Diurnal Variations in the Motility of Populations of Biflagellate Microalgae

The motility of microalgae has been studied extensively, particularly in model microorganisms such as Chlamydomonas reinhardtii. For this and other microalgal species, diurnal cycles are well known to control the metabolism, growth, and cell division. Diurnal variations, however, have been largely neglected in quantitative studies of motility. Here, we demonstrate using tracking microscopy how the motility statistics of C. reinhardtii are modulated by diurnal cycles. With nine independently inoculated cultures synchronized to the light-dark cycle at the exponential growth phase, we repeatedly observed that the mean swimming speed is greater during the dark period of a diurnal cycle. From this measurement, using a hydrodynamic power balance, we infer the mean flagellar beat frequency and conjecture that its diurnal variation reflects modulation of intracellular ATP. Our measurements also quantify the diurnal variations of the orientational and gravitactic transport of C. reinhardtii. We use this to explore the population-level consequences of diurnal variations of motility statistics by evaluating a prediction for how the gravitactic steady state changes with time during a diurnal cycle. Finally, we discuss the consequences of diurnal variations of microalgal motility in soil and pelagic environments.     

PHOTOTAXIS IN CHLAMYDOMONAS REINHARDTII

Parameters which distinguish phototaxis from random motility in Chlamydomonas reinhardtii have been defined with quantitative assays. The phototactic responses in photosynthetic, mixotrophic, and heterotrophic cultures were highest during exponential growth and declined rapidly as the cultures entered stationary phase. In contrast, random motility was relatively constant throughout growth. Phototaxis and motility also differ in their sensitivity to azide and antimycin A. Both of these drugs inhibited phototaxis within 5 min, but motility was unaffected for at least 30 min. Phototaxis and motility have different ion requirements. Optimum motility was observed in the presence of either Ca⁺⁺ or Mg⁺⁺; phototaxis required Ca⁺⁺ and either K⁺ or NH₄⁺. Photosynthesis is not required for phototaxis, since phototaxis was not inhibited by dichlorophenyldimethyl urea, and a mutant lacking chlorophyll was phototactic.     

Aerobic and anaerobic swimming speeds of spermatozoa investigated by twin beam laser velocimetry.

The motility of bovine and ovine spermatozoa has been studied under aerobic and anaerobic conditions, using a dual beam laser velocimeter. Cells swimming under aerobic conditions were found to be characterized by a translational swimming speed and a rotation rate that were approximately double those of cells swimming in an anaerobic environment. Both types of spermatozoa have been found to exhibit a sudden coordinated transition between fast and slow swimming states when the available oxygen is exhausted. This transition from aerobic to anaerobic swimming states has also been shown to be reversible. Studies of the duration of aerobic motility using the same apparatus have shown that the cells have a constant motile efficiency over the temperature range 32 degrees-42 degrees C.     

Stimulation of motility in cultured bovine capillary endothelial cells by angiogenic preparations

Several angiogenic preparations that have been shown to stimulate plasminogen activator (PA) and collagenase production by cultured bovine capillary endothelial (BCE) cells were tested for their ability to stimulate BCE cell motility in the phagokinetic track assay. Bovine retinal extract, medium conditioned by 3T3-F442A differentiated mouse adipocytes, SK HEP-1 human hepatoma cell lysate, mouse sarcoma 180 cell lysate, and medium conditioned by mouse sarcoma 180 cells stimulated motility 68.7%, 48.5%, 140.9%, 56.5%, and 102.1%, respectively, relative to untreated cells. The motility-stimulating activity of these preparations was dose dependent and linear over the 16-h assay period. Several hormones and growth factors were tested for BCE cell motility-stimulating activity, including insulin, vasopressin, fibroblast growth factor, and a partially purified preparation of sarcoma growth factor, and were found to be ineffective. 12-0-tetradecanoyl-phorbol-acetate (TPA), a potent stimulator of both PA and collagenase activities in BCE cells, also did not stimulate motility, indicating that protease production is not sufficient to stimulate BCE cell motility in this assay. Neither SK HEP-1 hepatoma cell lysate nor TPA was effective in stimulating motility in bovine aortic endothelial (BAE) cells. The inability of SK HEP-1 hepatoma cell lysate to stimulate movement in BAE cells is consistent with the observation that angiogenesis occurs by sprouting of capillaries, not large vessels.