ABSTRACT
Plasmodium sporozoite motility is essential for establishing malaria infections. It depends on initial adhesion to a substrate as well as the continuous turnover of discrete adhesion sites. Adhesion and motility are mediated by a dynamic actin cytoskeleton and surface proteins. The mode of adhesion formation and the integration of adhesion forces into fast and continuous forward locomotion remain largely unknown. Here, we use optical tweezers to directly trap individual parasites and probe adhesion formation. We find that sporozoites lacking the surface proteins TRAP and S6 display distinct defects in initial adhesion; trap(-) sporozoites adhere preferentially with their front end, while s6(-) sporozoites show no such preference. The cohesive strength of the initial adhesion site is differently affected by actin filament depolymerization at distinct adhesion sites along the parasite for trap(-) and s6(-) sporozoites. These spatial differences between TRAP and S6 in their functional interaction with actin filaments show that these proteins have nonredundant roles during adhesion and motility. We suggest that complex protein-protein interactions and signaling events govern the regulation of parasite gliding at different sites along the parasite. Investigating how these events are coordinated will be essential for our understanding of sporozoite gliding motility, which is crucial for malaria infection. Laser tweezers will be a valuable part of the toolset.
Subject(s)
Actins/chemistry , Cell Adhesion Molecules/chemistry , Optical Tweezers , Plasmodium falciparum/chemistry , Protein Interaction Mapping/methods , Protozoan Proteins/chemistry , Adhesiveness , Binding Sites , Protein BindingABSTRACT
Bundles of filamentous actin are dominant cytoskeletal structures, which play a crucial role in various cellular processes. As yet quantifying the fundamental interaction between two individual actin filaments forming the smallest possible bundle has not been realized. Applying holographic optical tweezers integrated with a microfluidic platform, we were able to measure the forces between two actin filaments during bundle formation. Quantitative analysis yields forces up to 0.2 pN depending on the concentration of bundling agents.
Subject(s)
Actins/chemistry , Biophysics/methods , Adenosine Triphosphate/chemistry , Animals , Cytoskeleton/metabolism , Electrolytes , Humans , Ions , Microfluidics , Nematoda , Optical Tweezers , Optics and Photonics , Reproducibility of Results , Stress, MechanicalABSTRACT
Holographic optical tweezers (HOT) are a versatile technology, with which complex arrays and movements of optical traps can be realized to manipulate multiple microparticles in parallel and to measure the forces affecting them in the piconewton range. We report on the combination of HOT with a fluorescence microscope and a stop-flow, multi-channel microfluidic device. The integration of a high-speed camera into the setup allows for the calibration of all the traps simultaneously both using Boltzmann statistics or the power spectrum density of the particle diffusion within the optical traps. This setup permits complete spatial, chemical and visual control of the microenvironment applicable to probing chemo-mechanical properties of cellular or subcellular structures. As an example we constructed a biomimetic, quasi-two-dimensional actin network on an array of trapped polystyrene microspheres inside the microfluidic chamber. During crosslinking of the actin filaments by Mg(2+) ions, we observe the build up of mechanical tension throughout the actin network. Thus, we demonstrate how our integrated HOT-microfluidics platform can be used as a reconfigurable force sensor array with piconewton resolution to investigate chemo-mechanical processes.
Subject(s)
Holography/instrumentation , Microfluidics/instrumentation , Optical Tweezers , Actins/chemistry , Actins/physiology , Algorithms , Biomimetic Materials , Calibration , Magnesium/chemistry , Microscopy, Fluorescence , Nanoparticles , Nanotechnology , Surface PropertiesABSTRACT
The cytoskeleton is a complex polymer network that plays an essential role in the functionality of eukaryotic cells. It endows cells with mechanical stability, adaptability, and motility. To identify and understand the mechanisms underlying this large variety of capabilities and to possibly transfer them to engineered networks makes it necessary to have in vitro and in silico model systems of the cytoskeleton. These models must be realistic representatives of the cellular network and at the same time be controllable and reproducible. Here, an approach to design complementary experimental and numerical model systems of the actin cytoskeleton is presented and some of their properties discussed.
Subject(s)
Actins/chemistry , Biomimetic Materials/chemistry , Cytoskeleton/chemistry , Models, Biological , Actins/ultrastructure , Animals , Cross-Linking Reagents , Cytoskeleton/ultrastructure , Dimethylpolysiloxanes/chemistry , Holography , Models, Molecular , Optical Tweezers , Polyethylene Glycols/chemistry , Rabbits , Shear StrengthABSTRACT
We introduce a method to tune the local orbital angular momentum density in an optical vortex beam without changing its topological charge or geometric intensity distribution. We show that adjusting the relative amplitudes a and b of two interfering collinear vortex beams of equal but opposite helicity provides the smooth variation of the orbital angular momentum density in the resultant vortex beam. Despite the azimuthal intensity modulations that arise from the interference, the local orbital angular momentum remains constant on the vortex annulus and scales with the modulation parameter, c = (a-b)/(a+b).
