Abstract
BackgroundThe field of structural dynamics of cytoskeletons in living cells is gathering wide interest, since better understanding of cytoskeleton intracellular organization will provide us with not only insights into basic cell biology but may also enable development of new strategies in regenerative medicine and cancer therapy, fields in which cytoskeleton-dependent dynamics play a pivotal role. The nanoneedle technology is a powerful tool allowing for intracellular investigations, as it can be directly inserted into live cells by penetrating through the plasma membrane causing minimal damage to cells, under the precise manipulation using atomic force microscope. Modifications of the nanoneedles using antibodies have allowed for accurate mechanical detection of various cytoskeletal components, including actin, microtubules and intermediate filaments. However, successful penetration of the nanoneedle through the plasma membrane has been shown to vary greatly between different cell types and conditions. In an effort to overcome this problem and improve the success rate of nanoneedle insertion into the live cells, we have focused here on the fluidity of the membrane lipid bilayer, which may hinder nanoneedle penetration into the cytosolic environment.ResultsWe aimed to reduce apparent fluidity of the membrane by either increasing the approach velocity or reducing experimental temperatures. Although changes in approach velocity did not have much effect, lowering the temperature was found to greatly improve the detection of unbinding forces, suggesting that alteration in the plasma membrane fluidity led to increase in nanoneedle penetration.ConclusionsOperation at a lower temperature of 4 °C greatly improved the success rate of nanoneedle insertion to live cells at an optimized approach velocity, while it did not affect the binding of antibodies immobilized on the nanoneedle to vimentins for mechanical detection. As these experimental parameters can be applied to various cell types, these results may improve the versatility of the nanoneedle technology to other cell lines and platforms.Electronic supplementary materialThe online version of this article (doi:10.1186/s12951-016-0226-5) contains supplementary material, which is available to authorized users.
Highlights
The field of structural dynamics of cytoskeletons in living cells is gathering wide interest, since better understanding of cytoskeleton intracellular organization will provide us with insights into basic cell biology but may enable development of new strategies in regenerative medicine and cancer therapy, fields in which cytoskeleton-dependent dynamics play a pivotal role
Development of nanoneedle-based techniques can lead to establishing of new technologies for mechanical analysis of the dynamic structure of cytoskeletal proteins in vivo and identification of the relevant proteins, in addition for its applications in cell sorting by the direct detection of the cytoskeleton
Evaluation of nanoneedle penetration into live cells Insertion of an atomic force microscopy (AFM) cantilever-type nanoneedle into the cytosol of live cells has been originally observed during AFM force spectroscopy measurements, evident from a sharp force relaxation peak in the force-distance curve during cell indentation
Summary
The field of structural dynamics of cytoskeletons in living cells is gathering wide interest, since better understanding of cytoskeleton intracellular organization will provide us with insights into basic cell biology but may enable development of new strategies in regenerative medicine and cancer therapy, fields in which cytoskeleton-dependent dynamics play a pivotal role. We have been developing a method for intracellular diagnosis of target cells by inserting a monolithic nanoneedle under the control of an atomic force microscopy (AFM) system [10,11,12,13] This nanoneedle technique allows mechanical detection of target molecules using force spectroscopy, a unique approach for detecting intracellular molecules in real-time [13,14,15]. An ultra-thin rod-shape with high-aspect ratio (Fig. 1a) allows for efficient insertion through the plasma membrane and into the cytosol of various cell types with minimal cell damage, while modification to the nanoneedle with antibodies allows for specific binding of the nanoneedle to intracellular cytoskeletal protein targets that can be quantified during needle evacuation from the cell. Optimizing insertion rates will significantly improve the future applicability of this technology [12, 17]
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