In general, cells show the ability to migrate in complex environments. Their direction of motion is often determined by external stimuli like gradients of chemo-attractants or topographical cues....Show moreIn general, cells show the ability to migrate in complex environments. Their direction of motion is often determined by external stimuli like gradients of chemo-attractants or topographical cues. The actin cortex plays an important role in the movement of crawling cell types. Previous research shows that actin is polymerized in waves[1]. The main goal of this project was to image and to design the analysis of these actin-polymerization waves and the influence of topographically constrains on these actin waves. Therefore, three different topographically constrained junctions were constructed. The result is a protocol to image highly motile amoeboid cells rearranging their actin cortex and the linkage with their global migrational behaviour. Spatio-temporal image correlation spectroscopy is advised for analyis of the actin-polymerization waves, due its high precision.Show less
Different rigidities have shown to be of effect on cell migration. The persistence in random cell motion is shown to be influenced by the rigidity of the substrate. We use various techniques to...Show moreDifferent rigidities have shown to be of effect on cell migration. The persistence in random cell motion is shown to be influenced by the rigidity of the substrate. We use various techniques to fabricate polydimethylsiloxane (PDMS) samples with a stiffness gradient, aiming at the biologically relevant, soft regime. Both using a temperature gradient and placing a UV irradiance profile on the PDMS substrate have been used to generate a stiffness gradient in PDMS. Highly motile amoeboid cells are placed on these substrates and their centre of mass motion is tracked using fluorescent tags. We used tools from statistical physics to analyze the nature of their motion, primarily focusing on the change in persistence over the tiffness gradient. We found that the persistence time of Dictyostelium discoideum is higher for stiffer uniform PMDS substrates, which substantiates the theory that the persistence time is related to durotaxis.Show less
It is recently discovered that not only chemicals are involved in cell movement. Also the mechanical surroundings play a big part in this. But how mechanical surroundings and cell movement are...Show moreIt is recently discovered that not only chemicals are involved in cell movement. Also the mechanical surroundings play a big part in this. But how mechanical surroundings and cell movement are coupled remains unexplained. A recent theoretical article by Novikova et al.[1] tries to explain this by introducing a coupling mechanism between the persistence time and the substrate rigidity for durotacting cells. By making three kinds of PDMS substrates and looking with fluorescence microscopy at the cell motion, we determine the persistence time and velocity of migrating Dd cells. First we looked at two homogeneous plates of PDMS and found a coupling between the rigidity of the PDMS substrate and the persistence time of the Dd cells that was opposite from what was claimed in [1]. We also looked at cell motion on a PDMS substrate stiffness gradient. Characterisation of stiffness gradients is different than characterisation of homogeneous plates, and is achieved by looking at the Durotactic Index, DI. Through this method, evidence is found for durotactic motion on a substrate stiffness gradient. Keywords: cell migration, PRW, durotaxis, gradientShow less
Cell migration plays a central role in keeping sturdy bones, the development of human embryos, the healing of wounds and in developing the blood circulation system. Fibroblasts is a type of cells...Show moreCell migration plays a central role in keeping sturdy bones, the development of human embryos, the healing of wounds and in developing the blood circulation system. Fibroblasts is a type of cells that also partake in migratory behavior. They do so in the healing process of skin tissue. Chemical stimuli play an important role in the migrating behavior of cells. To research the migratory behavior of 3T3 fibroblast cells by to the chemical stimulus of Epidermic Growth Factor (EGF), their chemokinetic and chemotactic reaction to the protein was tested. A chemokinetic reaction increases cell motility and is evoked in cells after exposing them to a concentration of a chemical stimulus. Average migration speed, a way to quantify the chemokinetic effect on cells, was studied in fibroblast after exposing them to different concentrations of EGF. They showed increased average migration speed at 10, 100 and 500 ng/ml EGF. The velocity difference between 100 ng/ml and 500 ng/ml proved to be negligible. For study of the chemotactic reaction of cells to a gradient of EGF, a triple inlet microfluidic flowchamber (MF), in which a gradient can be created, was characterized studying the diffusion of FITC-Dextran inside the MF. FITC-Dextran shares its diffusive properties with EGF. Once the diffusion of Dextran in a triplet inlet MF was studied, this would inform us about the manner in which EGF diffuses in the same MF. We found a gradient at 10 mm from the inlet of 25 µM / mm, ranging over 1000 to 1800 µm along the cross section. In the future, cells will be placed inside this well controlled gradient generator.Show less
Intracellular transport is the movement of cell elements throughout the cell, typically via passive diffusion or active transport. It is vital for the continued development, functioning and...Show moreIntracellular transport is the movement of cell elements throughout the cell, typically via passive diffusion or active transport. It is vital for the continued development, functioning and survival of cells. During active transport, components such as vesicles and particular organelles are transported to their destinations, using parts of the cytoskeleton as intracellular highways. This transport is carried out by motor proteins that attach to the cargoes and move them over the filaments and microtubules of the cell. In nerve cells active transport occurs not only in the cell body, but also in their neurites. Nerve cells are highly dependent on efficient intracellular transport within these neurites, as it is vital for their primary function of transmitting chemical signals to and from other cells. Impairment of this process can lead to reduced activity and even cell death. Cellular trafficking within the axons of several types of neurons has been found to be impaired in a variety of neurodegenerative diseases such as ALS, Alzheimer’s and Huntington’s. Investigation of the mechanics behind this impairment of transport would allow for a greater insight into the cause of these diseases, and possibly bring us closer to the development of an effective treatment. In this research project we used the PC12 cell line. These cells are a model system often used in neurobiological research as they exhibit many neuron-like properties, including neuronal differentiation. We quantified the movement of vesicles and mitochondria, both of which can be actively transported in the neurites. To reduce the number of parameters and facilitate verification through theoretical models we applied topographical and chemical guidance methods to direct the outgrowths of these neurites in one dimension. A time resolved mean squared displacement analysis was carried out on the trajectories of the vesicles and mitochondria to obtain information on the type of motion they undergo. The rate, type and speed of transport between the vesicles and mitochondria were compared. We found that the instantaneous velocities of vesicles both in active transport and diffusive motion are considerably higher than those of mitochondria. Further possible improvements and optimization of the experimental methods would lead to more significant statistical results, leading to insights into intracellular transport of the organelles in nerve cells.Show less
In the development and maintenance of multicellular organisms, cell migration is an essential process. It is therefore important to study the migration of cells. The analysis of the position of the...Show moreIn the development and maintenance of multicellular organisms, cell migration is an essential process. It is therefore important to study the migration of cells. The analysis of the position of the cell during migration is often done by tracking the center of mass of the cell in the case that it's impossible to track the position of the nucleus. This tracking point is not ideal since the formation of protrusions and retractions lead to a fluctuation in the position of this tracking point while the body may not actually be moving. By using the medial axis of the binary object corresponding to the cell, we found an alternative tracking point, which is a slight improvement over the center of mass as the tracking point. We succeeded in tracking and analyzing the protrusions formed by the cell. These findings open up the possibility to characterize different cell types by the behavior of their protrusions and to obtain a quantitative description of the molecules contributing to the formation and dynamics of protrusions.Show less
