During development, the axons of neurons in the mammalian central nervous system lose their ability to regenerate after injury. In order to study the regeneration process, we used a laser dissector system based on a sub-nanosecond pulsed UVA laser to inflict a partial damage to the axon of mouse hippocampal neurons. The use of such laser gives the possibility to deliver very low average power to the sample to be ablated. Therefore, the collateral damage due to temperature rise was reduced and for the first time, we were able to manipulate the neurite of cultured mouse neurons during the first days in vitro with sub-cellular precision. Force spectroscopy measurements were performed in parallel during and after the partial ablation of the neurite, by the use of a bead attached to the neurite membrane and held in an optical trap. The sub-piconewton and millisecond resolution of the force spectroscopy measurements allowed to quantify the damage inflicted to the process and to monitor the viscoelastic properties of the axonal membrane during regeneration. The reorganization and regeneration of the axon was documented by long-term (24-48 hours) bright-field live imaging using an optical microscope equipped with a custom-built cell culture incubator.
Articles tagged with: Force-clamp
Complex biological phenomena, such as cell differentiation and locomotion, require long range tracking capabilities with nanometer resolution over an extended period, to resolve molecular processes on the cellular scale.
Living cells face mechanical forces which are converted into biochemical signals and integrated into the cellular responses (mechanotransduction). Therefore, the development of new techniques for exerting mechanical stresses on cells and observe their responses is crucial to clarify the molecule‐and cell‐level structures that may participate in mechanotransduction.
Forces can be exerted on a cell by a variety of experimental techniques. If the force is in the right range of magnitude it is capable of eliciting a biological response from the cell. The magnitude of force that can be produced by a single myosin molecule8, consistent with the theory that active cellular contraction can induce cell signalling. Therefore, all this would suggest that the critical values of force exerted on a single molecule fall within the pN range. Interestingly to notice, this is also the range of forces exerted on the particle in an optical
trap by the radiation pressure of light. This relatively young technique provides the nonmechanical manipulation of the biological particles such as virus, living cells and subcellular organelles. Moreover, optical tweezers are recognized single-molecule technique to resolve forces and motion on the molecular scale.
Optical Tweezers are now being used in the investigation of an increasing number of biochemical and biophysical processes, from the mechanical properties of biological polymers to the multitude of molecular machines that drive the internal dynamics of the cell.
The combination of chemical and mechanical stimulation is useful and relevant in cell biology, since cells test the extracellular matrix rigidity during their differentiation.
Cells need to organize their internal space and interact with their environment. The network filaments, i.e., the cytoskeleton, represent connections to distribute tensile forces through the cytoplasm to nucleus as a signaling pathway. Transmembrane receptor, as integrins, function as mechanoreceptors and provide a preferred path to transfer mechanical forces across cell surface. During motion, cells organize integrins distribution in clusters at special complexes called focal adhesions. Forces transmitted to the cytoskeleton from these sites produce a stress in associated cytoskeleton molecules which can be converted in gene expression, protein recruitment at local sites and dynamic cytoskeleton architecture modulation. Thus cell shape and stability is the effect of a mechanical force balance between cytoskeleton traction forces and extracellular substrate local rigidity. Optical tweezers can be utilized to measure and analyze the force operated by cells in exploratory motion and therefore to understand how cell operate mechanic transduction. With optical microscopy, it is possible to follow the motion of the cell, but force
measurements permit to quantify how many molecular motors are involved in a movement and therefore to quantify how much the molecular machinery of the cell is recruited in such action. This means that thanks to force measurement, it is possible to distinguish random motion from well organized shift of the cell.
It has been observed that filopodia, which explore the environment by rapidly moving in all directions, modulate their activity changing the duration of the collision with a bead, in response to different stiffness of the load. This could represent sensing of the obstacle force and is in agreement with measurements on single microtubule catastrophic time modulation. Lamellipodia, which follow the pathway analyzed by filopodia, showed a more complex
behavior in response to the obstacle: sometimes, they entirely retracted, other times they moved around it to progress forward or they removed the obstacle by lifting it and giving it back. Lamellipodia have a more differentiated structure and are thought to exert a force with variable directions in space. Therefore, multi‐tweezers measurements have been applied to understand their overall organization and to measure complex forces exerted during growth cone motion.
This kind of measurements can be used to understand the response of the cells to molecular cues or mechanic‐chemical stimuli localized in the trapping volume, or to understand the mechanisms and which molecules are involved in different steps of motor planning. Moreover, when the system is working in force-clamp condition, can be exploited to study the role of statically and dynamically applied forces in neuronal differentiation.
During morphogenesis, neuronal precursor cells migrate from the zone where they are born to their final destination, which, in some cases, is at a distance of several millimeters. After reaching their destination, neurons must establish appropriate synaptic connections by sending out from their soma projections called neurites. The motion of neurites is guided by growth cones located at their tips. Growth cones contain a variety of chemical and
mechanical receptors and sophisticated biochemical machinery that couples these receptors to the cytoskeleton. Extruding from the tip of the growth cone are highly motile structures called filopodia and lamellipodia that are used to explore and probe the environment. All these complex events, which are at the basis of neuronal development and differentiation, involve cell motility requiring a precise control of cellular and molecular motors.
We study, by optical tweezers and force spectroscopy, the dynamic of cytoskeletal elements in the growth cone, and how the growth cone navigate in a controlled mechano-chemical micro-enviroment.