Confirmation and variability of the Allee effect inDictyostelium discoideumcell populations, possible role of chemical signaling within cell clusters.

In studies of the unicellular eukaryoteDictyostelium discoideum, many have anecdotally observed that cell dilution below a certain 'threshold density' causes cells to undergo a period of slow growth (lag). However, little is documented about the slow growth phase and the reason for different growth dynamics below and above this threshold density. In this paper, we extend and correct our earlier work to report an extensive set of experiments, including the use of new cell counting technology, that set this slow-to-fast growth transition on a much firmer biological basis. We show that dilution below a certain density (around 104cells ml-1) causes cells to grow slower on average and exhibit a large degree of variability: sometimes a sample does not lag at all, while sometimes it takes many moderate density cell cycle times to recover back to fast growth. We perform conditioned media experiments to demonstrate that a chemical signal mediates this endogenous phenomenon. Finally, we argue that while simple models involving fluid transport of signal molecules or cluster-based signaling explain typical behavior, they do not capture the high degree of variability between samples but nevertheless favor an intra-cluster mechanism.

Extracellular Processing of Molecular Gradients by Eukaryotic Cells Can Improve Gradient Detection Accuracy.

Eukaryotic cells sense molecular gradients by measuring spatial concentration variation through the difference in the number of occupied receptors to which molecules can bind. They also secrete enzymes that degrade these molecules, and it is presently not well understood how this affects the local gradient perceived by cells. Numerical and analytical results show that these enzymes can substantially increase the signal-to-noise ratio of the receptor difference and allow cells to respond to a much broader range of molecular concentrations and gradients than they would without these enzymes.

Spontaneous emergence of large-scale cell cycle synchronization in amoeba colonies.

Unicellular eukaryotic amoebae Dictyostelium discoideum are generally believed to grow in their vegetative state as single cells until starvation, when their collective aspect emerges and they differentiate to form a multicellular slime mold. While major efforts continue to be aimed at their starvation-induced social aspect, our understanding of population dynamics and cell cycle in the vegetative growth phase has remained incomplete. Here we show that cell populations grown on a substrate spontaneously synchronize their cell cycles within several hours. These collective population-wide cell cycle oscillations span millimeter length scales and can be completely suppressed by washing away putative cell-secreted signals, implying signaling by means of a diffusible growth factor or mitogen. These observations give strong evidence for collective proliferation behavior in the vegetative state.

High fidelity information processing in folic acid chemotaxis of Dictyostelium amoebae.

Living cells depend upon the detection of chemical signals for their existence. Eukaryotic cells can sense a concentration difference as low as a few per cent across their bodies. This process was previously suggested to be limited by the receptor-ligand binding fluctuations. Here, we first determine the chemotaxis response of Dictyostelium cells to static folic acid gradients and show that they can significantly exceed this sensitivity, responding to gradients as shallow as 0.2% across the cell body. Second, using a previously developed information theory framework, we compare the total information gained about the gradient (based on the cell response) to its upper limit: the information gained at the receptor-ligand binding step. We find that the model originally applied to cAMP sensing fails as demonstrated by the violation of the data processing inequality, i.e. the total information exceeds the information at the receptor-ligand binding step. We propose an extended model with multiple known receptor types and with cells allowed to perform several independent measurements of receptor occupancy. This does not violate the data processing inequality and implies the receptor-ligand binding noise dominates both for low- and high-chemoattractant concentrations. We also speculate that the interplay between exploration and exploitation is used as a strategy for accurate sensing of otherwise unmeasurable levels of a chemoattractant.

Live cell flattening - traditional and novel approaches.

Eukaryotic cell flattening is valuable for improving microscopic observations, ranging from bright field (BF) to total internal reflection fluorescence (TIRF) microscopy. Fundamental processes, such as mitosis and in vivo actin polymerization, have been investigated using these techniques. Here, we review the well known agar overlayer protocol and the oil overlay method. In addition, we present more elaborate microfluidics-based techniques that provide us with a greater level of control. We demonstrate these techniques on the social amoebae Dictyostelium discoideum, comparing the advantages and disadvantages of each method.PACS Codes: 87.64.-t, 47.61.-k, 87.80.Ek.

Contact-mediated cell-assisted cell proliferation in a model eukaryotic single-cell organism: an explanation for the lag phase in shaken cell culture.

In cell culture, when cells are inoculated into fresh media, there can be a period of slow (or lag phase) growth followed by a transition to exponential growth. This period of slow growth is usually attributed to the cells' adaptation to a new environment. However, we argue that, based on observations of shaken suspension culture of Dictyostelium discoideum, a model single-cell eukaryote, this transition is due to a density effect. Attempts to demonstrate the existence of implicit cell signaling via long-range diffusible messengers (i.e., soluble growth factors) through cell-medium separation and microfluidic flow perturbation experiments produced negative results. This, in turn, led to the development of a signaling model based on direct cell-to-cell contacts. Employing a scaling argument for the collision rate due to fluid shear, we reasonably estimate the crossover density for the transition into the exponential phase and fit the observed growth kinetics.

Dictyostelium discoideum chemotaxis: threshold for directed motion.

The chemotactic response of Dictyostelium discoideum cells to stationary, linear gradients of cyclic adenosine 3',5'-monophosphate (cAMP) was studied using microfluidic devices. In shallow gradients of less than 10(-3) nM/microm, the cells showed no directional response and exhibited a constant basal motility. In steeper gradients, cells moved up the gradient on average. The chemotactic speed and the motility increased with increasing steepness up to a plateau at around 10(-1) nM/microm. In very steep gradients, above 10 nM/microm, the cells lost directionality and the motility returned to the sub-threshold level. In the regime of optimal response the difference in receptor occupancy at the front and back of the cell is estimated to be only about 100 molecules.

Improving slide-based assays by stirring: application of liquid-on-liquid mixing to immunofluorescence staining of polytene chromosomes.

A new method for stirring thin liquid films has been developed and demonstrated to increase the sensitivity of immunofluorescence staining of polytene chromosomes. This liquid-on-liquid mixing (LOLM) technique uses a stirrer fluid, immiscible with the thin film, to transmit shear at the liquid-liquid interface. Here, we stir mineral oil layered over an aqueous thin film of antibody solution, which stains transcription apparatuses on chromosomes previously fixed to a glass slide. The quality of staining was assessed at varying antibody concentrations and incubation or stirring times. Our data indicate that the LOLM technique overcomes the diffusion barrier associated with traditional slide-based biological assays.

Pair correlations of a dilute charged colloidal fluid near a glass wall.

Using confocal microscopy we examine the static structure of low density, highly charged colloidal suspensions near a repulsive glass boundary. We find no sign of an interparticle attraction of the magnitude noted previously.