Differential Dynamic Microscopy: A High-Throughput Method for Characterizing the Motility of Microorganisms

We present a fast, high-throughput method for characterizing the motility of microorganisms in three dimensions based on standard imaging microscopy. Instead of tracking individual cells, we analyze the spatiotemporal fluctuations of the intensity in the sample from time-lapse images and obtain the intermediate scattering function of the system. We demonstrate our method on two different types of microorganisms: the bacterium Escherichia coli (both smooth swimming and wild type) and the biflagellate alga Chlamydomonas reinhardtii. We validate the methodology using computer simulations and particle tracking. From the intermediate scattering function, we are able to extract the swimming speed distribution, fraction of motile cells, and diffusivity for E. coli, and the swimming speed distribution, and amplitude and frequency of the oscillatory dynamics for C. reinhardtii. In both cases, the motility parameters were averaged over ∼ 10⁴ cells and obtained in a few minutes.     

Characterisation of Mytilus edulis hemocyte subpopulations by single cell time-lapse motility imaging

In bivalve molluscs, defence against pathogens mainly relies on fast tissue infiltration by immunocompetent hemocytes that migrate from circulating hemolymph to sites of infection, in order to deliver, in situ, an effective immune response. In the present work, we have investigated dynamics of hemocyte subpopulations motility by combining flow cytometry coupled to Coulter-type cell volume determination, Hoffman modulation contrast microscopy, time-lapse imaging and off-line analysis of cell shape changes. Our results revealed fast modifications of hemocyte aspect in vitro, with bidirectional transitions from spread outlines to condensed cell body morphologies, in the minute range. Amoeboid or non-amoeboid types of locomotion were observed, depending on the cell shapes and on the cell subtypes, with velocities reaching up to 30 μm min^?1. Correlations between motion profiles, Hemacolor staining and flow cytometry analysis on living cells help to propose a functional mussel hemocyte classification including the motile properties of these cells. In particular, basophils were shown to be involved in dynamic hemocyte–hemocyte interactions and in the constitution of aggregation cores. Physiological implications, in terms of immune response in organisms devoid of endothelium-closed vascular system, and potential applications of hemocyte motility studies for the development and the interpretation of experiments involving hemocytes in the field of marine ecotoxicology are discussed.     

THE ROLE OF THREE CYTOPLASMIC FIBERS IN BHK-21 CELL MOTILITY : I. Microtubules and the Effects of Colchicine

Microtubule breakdown in the presence of 5 or 40 µg/ml of colchicine is observed in BHK-21/C13 fibroblast-like cells. Several morphological and physiological effects are noted in the absence of microtubules: (a) the cells transform from fibroblast-like to epithelial-like cells; (b) the normal pattern of intracellular birefringence changes and a juxtanuclear cap of birefringent filaments is formed; (c) time-lapse cinematography demonstrates that cell locomotion is inhibited in colchicine-treated cells, even though membrane ruffling persists. The results are discussed in terms of the specific roles of microtubules in cultured cell motility and possible functional relationships of the three types of cytoplasmic fibers seen in BHK-21 cells.     

Matrix metalloproteinase 2-integrin alpha(v)beta3 binding is required for mesenchymal cell invasive activity but not epithelial locomotion: a computational time-lapse study

Cellular invasive behavior through three-dimensional collagen gels was analyzed using computational time-lapse imaging. A subpopulation of endocardial cells, derived from explanted quail cardiac cushions, undergoes an epithelial-to-mesenchymal transition and invades the substance of the collagen gels when placed in culture. In contrast, other endocardial cells remain epithelial and move over the gel surface. Here, we show that integrin αvβ3 and matrix metalloproteinase (MMP)2 are present and active in cushion mesenchymal tissue. More importantly, functional assays show that mesenchymal invasive behavior is dependent on MMP2 activity and integrin αvβ3 binding. Inhibitors of MMP enzymatic activity and molecules that prevent integrin αvβ3 binding to MMP2, via its hemopexin domain, result in significantly reduced cellular protrusive activity and invasive behavior. Computational analyses show diminished intensity and persistence time of motility in treated invasive mesenchymal cells, but no reduction in motility of the epithelial-like cells moving over the gel surface. Thus, quantitative time-lapse data show that mesenchymal cell invasive behavior, but not epithelial cell locomotion over the gel surface, is partially regulated by the MMP2–integrin interactions.     

Lactotransferrin in Asian Elephant (Elephas maximus) Seminal Plasma Correlates with Semen Quality

Asian elephants (Elephas maximus) have highly variable ejaculate quality within individuals, greatly reducing the efficacy of artificial insemination and making it difficult to devise a sperm cryopreservation protocol for this endangered species. Because seminal plasma influences sperm function and physiology, including sperm motility, the objectives of this study were to characterize the chemistry and protein profiles of Asian elephant seminal plasma and to determine the relationships between seminal plasma components and semen quality. Ejaculates exhibiting good sperm motility (≥65%) expressed higher percentages of spermatozoa with normal morphology (80.3±13.0 vs. 44.9±30.8%) and positive Spermac staining (51.9±14.5 vs. 7.5±14.4%), in addition to higher total volume (135.1±89.6 vs. 88.8±73.1 ml) and lower sperm concentration (473.0±511.2 vs. 1313.8±764.7×10⁶ cells ml⁻¹) compared to ejaculates exhibiting poor sperm motility (≤10%; P<0.05). Comparison of seminal plasma from ejaculates with good versus poor sperm motility revealed significant differences in concentrations of creatine phosphokinase, alanine aminotransferase, phosphorus, sodium, chloride, magnesium, and glucose. These observations suggest seminal plasma influences semen quality in elephants. One- and two-dimensional (2D) gel electrophoresis revealed largely similar compositional profiles of seminal plasma proteins between good and poor motility ejaculates. However, a protein of ∼80 kDa was abundant in 85% of ejaculates with good motility, and was absent in 90% of poor motility ejaculates (P<0.05). We used mass spectrometry to identify this protein as lactotransferrin, and immunoblot analysis to confirm this identification. Together, these findings lay a functional foundation for understanding the contributions of seminal plasma in the regulation of Asian elephant sperm motility, and for improving semen collection and storage in this endangered species.     

β-Actin Messenger RNA Localization and Protein Synthesis Augment Cell Motility

In chicken embryo fibroblasts (CEFs), β-actin mRNA localizes near an actin-rich region of cytoplasm specialized for motility, the lamellipodia. This localization is mediated by isoform-specific 3′-untranslated sequences (zipcodes) and can be inhibited by antizipcode oligodeoxynucleotides (ODNs) (Kislauskis, E.H., X.-C. Zhu, and R.H. Singer. 1994. J. Cell Biol. 127: 441–451). This inhibition of β-actin mRNA localization resulted in the disruption of fibroblast polarity and, presumably, cell motility. To investigate the role of β-actin mRNA in motility, we correlated time-lapse images of moving CEFs with the distribution of β-actin mRNA in these cells. CEFs with localized β-actin mRNA moved significantly further over the same time period than did CEFs with nonlocalized mRNA. Antizipcode ODN treatment reduced this cell translocation while control ODN treatments showed no effect. The temporal relationship of β-actin mRNA localization to cell translocation was investigated using serum addition to serum-deprived cultures. β-actin mRNA was not localized in serum-deprived cells but became localized within minutes after serum addition (Latham, V.M., E.H. Kislauskis, R.H. Singer, and A.F. Ross. 1994. J. Cell Biol. 126:1211–1219). Cell translocation increased over the next 90 min, and actin synthesis likewise increased. Puromycin reduced this cell translocation and blocked this induction in cytosolic actin content. The serum induction of cell movement was also inhibited by antizipcode ODNs. These observations support the hypothesis that β-actin mRNA localization and consequent protein synthesis augment cell motility.     

Disruption of TgPHIL1 Alters Specific Parameters of Toxoplasma gondii Motility Measured in a Quantitative,Three-Dimensional Live Motility Assay

T. gondii uses substrate-dependent gliding motility to invade cells of its hosts, egress from these cells at the end of its lytic cycle and disseminate through the host organism during infection. The ability of the parasite to move is therefore critical for its virulence. T. gondii engages in three distinct types of gliding motility on coated two-dimensional surfaces: twirling, circular gliding and helical gliding. We show here that motility in a three-dimensional Matrigel-based environment is strikingly different, in that all parasites move in irregular corkscrew-like trajectories. Methods developed for quantitative analysis of motility parameters along the smoothed trajectories demonstrate a complex but periodic pattern of motility with mean and maximum velocities of 0.58±0.07 µm/s and 2.01±0.17 µm/s, respectively. To test how a change in the parasite''s crescent shape might affect trajectory parameters, we compared the motility of Δphil1 parasites, which are shorter and wider than wild type, to the corresponding parental and complemented lines. Although comparable percentages of parasites were moving for all three lines, the Δphil1 mutant exhibited significantly decreased trajectory lengths and mean and maximum velocities compared to the parental parasite line. These effects were either partially or fully restored upon complementation of the Δphil1 mutant. These results show that alterations in morphology may have a significant impact on T. gondii motility in an extracellular matrix-like environment, provide a possible explanation for the decreased fitness of Δphil1 parasites in vivo, and demonstrate the utility of the quantitative three-dimensional assay for studying parasite motility.     

Motility of Colwellia psychrerythraea Strain 34H at Subzero Temperatures

We examined the Arctic bacterium Colwellia psychrerythraea strain 34H for motility at temperatures from −1 to −15°C by using transmitted-light microscopy in a temperature-controlled laboratory. The results, showing motility to −10°C, indicate much lower temperatures to be permissive of motility than previously reported (5°C), with implications for microbial activity in frozen environments.     

The Effect of Simulating Different Intermediate Host Snail Species on the Link between Water Temperature and Schistosomiasis Risk

Introduction

A number of studies have attempted to predict the effects of climate change on schistosomiasis risk. The importance of considering different species of intermediate host snails separately has never previously been explored.

Methods

An agent-based model of water temperature and Biomphalaria pfeifferi population dynamics and Schistosoma mansoni transmission was parameterised to two additional species of snail: B. glabrata and B. alexandrina.

Results

Simulated B. alexandrina populations had lower minimum and maximum temperatures for survival than B. pfeifferi populations (12.5–29.5°C vs. 14.0–31.5°C). B. glabrata populations survived over a smaller range of temperatures than either B. pfeifferi or B. alexandrina (17.0°C–29.5°C). Infection risk peaked at 16.5°C, 25.0°C and 19.0°C respectively when B. pfeifferi, B. glabrata and B. alexandrina were simulated. For all species, infection risk increased sharply once a minimum temperature was reached.

Conclusions

The results from all three species suggest that infection risk may increase dramatically with small increases in temperature in areas at or near the currents limits of schistosome transmission. The effect of small increases in temperature in areas where schistosomiasis is currently found will depend both on current temperatures and on the species of snail acting as intermediate host(s) in the area. In most areas where B. pfeifferi is the host, infection risk is likely to decrease. In cooler areas where B. glabrata is the host, infection risk may increase slightly. In cooler areas where B. alexandrina is the host, infection risk may more than double with only 2°C increase in temperature. Our results show that it is crucial to consider the species of intermediate host when attempting to predict the effects of climate change on schistosomiasis.     

Assessment of the Roles of LuxS, S-Ribosyl Homocysteine, and Autoinducer 2 in Cell Attachment during Biofilm Formation by Listeria monocytogenes EGD-e

LuxS is responsible for the production of autoinducer 2 (AI-2), which is involved in the quorum-sensing response of Vibrio harveyi. AI-2 is found in several other gram-negative and gram-positive bacteria and is therefore considered a good candidate for an interspecies communication signal molecule. In order to determine if this system is functional in the gastrointestinal pathogen Listeria monocytogenes EGD-e, an AI-2 bioassay was performed with culture supernatants. The results indicated that this bacterium produces AI-2 like molecules. A potential ortholog of V. harveyi luxS, lmo1288, was found by performing sequence similarity searches and complementation experiments with Escherichia coli DH5α, a luxS null strain. lmo1288 was found to be a functional luxS ortholog involved in AI-2 synthesis. Indeed, interruption of lmo1288 resulted in loss of the AI-2 signal. Although no significant differences were observed between Lux1 and EGD-e with regard to planktonic growth (at 10°C, 15°C, 25°C, and 42°C), swimming motility, and phospholipase and hemolytic activity, biofilm culture experiments showed that under batch conditions between 25% and 58% more Lux1 cells than EGD-e cells were attached to the surface depending on the incubation time. During biofilm growth in continuous conditions after 48 h of culture, Lux1 biofilms were 17 times denser than EGD-e biofilms. Finally, our results showed that Lux1 accumulates more S-adenosyl homocysteine (SAH) and S-ribosyl homocysteine (SRH) in culture supernatant than the parental strain accumulates and that SRH, but not SAH or AI-2, is able to modify the number of attached cells.     

The Tetraspanins CD9 and CD81 Regulate CD9P1-Induced Effects on Cell Migration

CD9P-1 is a cell surface protein with immunoglobulin domains and an unknown function that specifically associates with tetraspanins CD9 and CD81. Overexpression of CD9P-1 in HEK-293 cells induces dramatic changes in cell spreading and migration on various matrices. Experiments using time-lapse videomicroscopy revealed that CD9P-1 expression has led to higher cell motility on collagen I but lower motility on fibronectin through a β1-integrins dependent mechanism. On collagen I, the increase in cell motility induced by CD9P-1 expression was found to involve integrin α2β1 and CD9P-1 was observed to associate with this collagen receptor. The generation of CD9P-1 mutants demonstrated that the transmembrane and the cytoplasmic domains are necessary for inducing effects on cell motility. On the other hand, expression of tetraspanins CD9 or CD81 was shown to reverse the effects of CD9P-1 on cell motility on collagen I or fibronectin with a concomitant association with CD9P-1. Thus, the ratio of expression levels between CD9P-1 and its tetraspanin partners can regulate cell motility.     

Erythrina mulungu Alkaloids Are Potent Inhibitors of Neuronal Nicotinic Receptor Currents in Mammalian Cells

Crude extracts and three isolated alkaloids from Erythrina mulungu plants have shown anxiolytic effects in different animal models. We investigated whether these alkaloids could affect nicotinic acetylcholine receptors and if they are selective for different central nervous system (CNS) subtypes. Screening experiments were performed using a single concentration of the alkaloid co-applied with acetylcholine in whole cell patch-clamp recordings in three different cell models: (i) PC12 cells natively expressing α3* nicotinic acetylcholine receptors; (ii) cultured hippocampal neurons natively expressing α7* nicotinic acetylcholine receptors; and (iii) HEK 293 cells heterologoulsy expressing α4β2 nicotinic acetylcholine receptors. For all three receptors, the percent inhibition of acetylcholine-activated currents by (+)-11á-hydroxyerysotrine was the lowest, whereas (+)-erythravine and (+)-11á-hydroxyerythravine inhibited the currents to a greater extent. For the latter two substances, we obtained concentration-response curves with a pre-application protocol for the α7* and α4β2 nicotinic acetylcholine receptors. The IC₅₀ obtained with (+)-erythravine and (+)-11á-hydroxyerythravine were 6 µM and 5 µM for the α7* receptors, and 13 nM and 4 nM for the α4β2 receptors, respectively. Our data suggest that these Erythrina alkaloids may exert their behavioral effects through inhibition of CNS nicotinic acetylcholine receptors, particularly the α4β2 subtype.     

Effects of Cholesterol-Loaded Cyclodextrins on the Rate and the Quality of Motility in Frozen and Thawed Rabbit Sperm

The motility of sperm after freezing and thawing is critical for effective cryopreservation. It is known that supplementation with cholesterol-loaded cyclodextrin (CLC) improves cryosurvival of sperm in various animals. To clarify the effects of supplementation with CLC on rabbit sperm motility after freezing and thawing, rabbit sperm motility was analyzed using a computer-assisted sperm analysis system. Sperm motility with CLC supplementation was 29.4 ± 9.6% (mean ± SD), which was significantly higher than that of controls (20.8 ± 7.1%, P<0.05). The curvilinear velocity of sperm with CLC exceeded that of controls, whereas the values for linearity and wobble were significantly lower in sperm with CLC compared with controls. After artificial insemination, 44.3% of recovered ova were fertilized in the CLC-supplemented group, which was higher than the percentage in the control group (36.4%). The results indicate that supplementation with CLC improves the rate and quality of motility in rabbit sperm after freezing and thawing, and would be advantageous for successful cryopreservation.     

Extracellular Heat Shock Protein 90 Signals through Subdomain II and the NPVY Motif of LRP-1 Receptor to Akt1 and Akt2: a Circuit Essential for Promoting Skin Cell Migration In Vitro and Wound Healing In Vivo

Normal cells secrete heat shock protein 90 alpha (Hsp90α) in response to tissue injury. Tumor cells have managed to constitutively secrete Hsp90α during invasion and metastasis. The sole function of extracellular Hsp90α (eHsp90α) is to promote cell motility, a critical event for both wound healing and tumor progression. The mechanism of promotility action by eHsp90α, however, has remained elusive. A key issue is whether eHsp90α still acts as a chaperone outside the cells or is a new and bona fide signaling molecule. Here, we have provided evidence that eHsp90α utilizes a unique transmembrane signaling mechanism to promote cell motility and wound healing. First, subdomain II in the extracellular part of low-density lipoprotein receptor-related protein 1 (LRP-1) receives the eHsp90α signal. Then, the NPVY but not the NPTY motif in the cytoplasmic tail of LRP-1 connects eHsp90α signaling to serine 473 but not threonine 308 phosphorylation in Akt kinases. Individual knockdown of Akt1, Akt2, or Akt3 revealed the importance of Akt1 and Akt2 in eHsp90α-induced cell motility. Akt gene rescue experiments suggest that Akt1 and Akt2 work in concert, rather than independently, to mediate eHsp90α promotility signaling. Finally, Akt1 and Akt2 knockout mice showed impaired wound healing that cannot be corrected by topical application with the eHsp90α protein.     

Influence of antibiotics on bacterial load and sperm parameters during short-term preservation of collared peccary semen

Studies on semen and sperm cells are critical to develop assisted reproductive technologies for the conservation of the collared peccary. The objective of the study was to compare the effect of different antibiotics on the bacterial load and sperm quality during short-term storage of peccary semen. Fresh semen samples from 10 males were extended in Tris-egg yolk or Tris-Aloe vera supplemented with streptomycin-penicillin (SP; 1 mg/mL - 1000 IU/mL or 2 mg/mL - 2000 IU/mL) or gentamicin (30 µg/mL or 70 µg/mL) before storage at 5°C. Bacterial load and sperm motility, membrane integrity and function, mitochondrial activity, and morphology, were evaluated at different time points for 36 h. The SP and gentamicin treatments concentration inhibited (p < 0.05) bacterial growth for 36 h regardless of the extender. Compared to the other treatments, Tris-egg yolk plus 70 µg/mL gentamicin maintained the sperm parameters for longer, including total motility (41.9 ± 6.1%) at 24 h, and membrane integrity (58.3 ± 2.1%) at 36 h. In contrast, the highest SP concentration in both extenders impaired sperm membrane integrity at 36 h (p < 0.05). For the liquid storage of collared peccary semen, it therefore is recommended to use Tris extender supplemented with egg yolk and gentamicin (70 µg/mL).     

Kinetic Cell-Cycle Analysis of a Cultured Mammalian Cell Population

The parameters of the cell cycle are analyzed in terms of the stochastic theory of cell proliferation for a murine mastocytoma line. The cells were grown in suspension culture under steady-state conditions in a chemostat. Initial estimates of the parameters from synchronous growth indicate that agreement of the data with the model is obtained only if the model is modified to include an initial proliferating fraction of less than 100%, and a cell loss continuing throughout the course of the experiment. The analysis verifies that the modified theory adequately describes the data, and that similar parameters are obtained from both desynchronization and percent labeled mitosis experiments. The average cycle time from 10 desynchronization experiments was 8.24 ± 0.52 h with a cellular standard deviation of 1.28 ± 0.18. The combined parameter obtained by dividing the cellular standard deviation by the cycle time is shown to be a useful measure of biological variability well defined over many different experiments. The rate constant for cell loss is about 0.009 which gives an 8% cell loss per cycle. The cell loss is sufficient to account for the apparent deficit in initially proliferating cells. The initial distribution of the synchronous cells is qualitatively examined and is found to be peaked late in G₁ or early in S.     

Automated time-lapse microscopy and high-resolution tracking of cell migration

We describe a novel fully automated high-throughput time-lapse microscopy system and evaluate its performance for precisely tracking the motility of several glioma and osteoblastic cell lines. Use of this system revealed cell motility behavior not discernable with conventional techniques by collecting data (1) from closely spaced time points (minutes), (2) over long periods (hours to days), (3) from multiple areas of interest, (4) in parallel under several different experimental conditions. Quantitation of true individual and average cell velocity and path length was obtained with high spatial and temporal resolution in “scratch” or “wound healing” assays. This revealed unique motility dynamics of drug-treated and adhesion molecule-transfected cells and, thus, this is a considerable improvement over current methods of measurement and analysis. Several fluorescent vital labeling methods commonly used for end-point analyses (GFP expression, DiO lipophilic dye, and Qtracker nanocrystals) were found to be useful for time-lapse studies under specific conditions that are described. To illustrate one application, fluorescently labeled tumor cells were seeded onto cell monolayers expressing ectopic adhesion molecules, and this resulted in consistently reduced tumor cell migration velocities. These highly quantitative time-lapse analysis methods will promote the creation of new cell motility assays and increase the resolution and accuracy of existing assays.Joseph S. Fotos and Vivek P. Patel contributed equally to this work     

Development of a novel rapid assay to assess the fidelity of DNA double-strand-break repair in human tumour cells

Cellular survival following ionising radiation-mediated damage is primarily a function of the ability to successfully detect and repair DNA double-strand breaks (DSBs). Previous studies have demonstrated that radiosensitivity, determined as a reduction in colony forming ability in vitro, may be related to the incorrect repair (misrepair) of DSBs. The novel rapid dual fluorescence (RDF) assay is a plasmid-based reporter system that rapidly assesses the correct rejoining of a restriction-enzyme produced DSBs within transfected cells. We have utilised this novel assay to determine the fidelity of DSB repair in the prostate tumour cell line LNCaP, the bladder tumour cell line MGH-U1 and a radiosensitive subclone S40b. The two bladder cell lines have been shown in previous studies to differ in their ability to correctly repair plasmids containing a single DSB. Using the RDF assay we found that a substantial portion of LNCaP cells [80.4 ± 5.3(standard error)%] failed to reconstitute reporter gene expression; however, there was little difference in this measure of DSB repair fidelity between the two bladder cell lines (48.3 ± 3.5% for MGH-U1; 39.9 ± 8.2% for S40b). The RDF assay has potential to be developed to study the relationship between DSB repair fidelity and radiosensitivity as well as the mechanisms associated with this type of repair defect.     

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

In vertebrates, hematopoiesis is regulated by inductive microenvironments (niches). Likewise, in the invertebrate model organism Drosophila melanogaster, inductive microenvironments known as larval Hematopoietic Pockets (HPs) have been identified as anatomical sites for the development and regulation of blood cells (hemocytes), in particular of the self-renewing macrophage lineage. HPs are segmentally repeated pockets between the epidermis and muscle layers of the larva, which also comprise sensory neurons of the peripheral nervous system. In the larva, resident (sessile) hemocytes are exposed to anti-apoptotic, adhesive and proliferative cues from these sensory neurons and potentially other components of the HPs, such as the lining muscle and epithelial layers. During normal development, gradual release of resident hemocytes from the HPs fuels the population of circulating hemocytes, which culminates in the release of most of the resident hemocytes at the beginning of metamorphosis. Immune assaults, physical injury or mechanical disturbance trigger the premature release of resident hemocytes into circulation. The switch of larval hemocytes between resident locations and circulation raises the need for a common standard/procedure to selectively isolate and quantify these two populations of blood cells from single Drosophila larvae. Accordingly, this protocol describes an automated method to release and quantify the resident and circulating hemocytes from single larvae. The method facilitates ex vivo approaches, and may be adapted to serve a variety of developmental stages of Drosophila and other invertebrate organisms.