Presentation Information
[P03-345]Microfluidic cell unroofing for the in situ analysis of organelle molecules without membrane permeabilization
○Yuki Umeda1, Shinya Yamahira1,2, Akimitsu Okamoto3, Satoshi Yamaguchi1,2 (1. SANKEN, Osaka University (Japan), 2. IIS, The University of Tokyo (Japan), 3. School of Engineering, The University of Tokyo (Japan))
Keywords:
Cell unroofing,Organelle,Microfluidics,Immunostaining,Membrane proteins
[Purpose]
Molecular networks of organelle membranes are involved in many cell processes. However, the nature of plasma membrane as a barrier to various analytical tools, including antibodies, makes it challenging to examine intact organelle membranes without affecting their structure and functions via membrane permeabilization. For now, to overcome the problem some "cell unroofing" methods have been developed, in which cells are fractured with sonication probe and so on, and intracellular membrane surface is exposed. However, those methods examined only over intracellular surface of plasma membrane and but over organelle membrane surface, due to the loss of whole intracellular compartment under unroofing treatment. Therefore, in this study, we aimed to develop a microfluidic method to unroof cells and observe the intrinsic membrane molecules in organelles.
[Method]
In our method, single cells were precisely arrayed on the bottom surface of microchannels in a light-guided manner using a photoactivatable cell anchoring material. Then, induced fast laminar flax was hit on cells and physically some part of them was flowed away. However, this stress on cells can be controled by cell intervals. In detail, when cell intervals were long, flax hit onto the side of cells and most cell compartments without bottom plasma membrane were removed. On the other hand, at sufficiently short cell intervals, horizontal stresses generated by the laminar flow cannot approach the cell side and instantly fractured only the upper cell membranes, without significantly affecting some organelles inside the fractured cells. This was also supported by computational fluid dynamics simulation of flow around cells.
[Results and Consideration]
Subsequently, left compartment in unroofed cells were observed via confocal fluorescence and scanning electron microscopy. Confocal observation showed the loss of apical cell membrane and remained calcein-stained cytoplasmic compartment. Moreover, electron microscopy suggested the existence of some kinds of organelles including nucleus and mitochondria. Next, the healthiness of left organelle was confirmed through the detection of membrane potential, which is an indicator for damage on mitochondria. As a result, unroofed cells were suggested to sustain the membrane potential and to be protected from significant damage. Furthermore, distribution of the mitochondrial membrane protein, translocase of outer mitochondrial membrane 20, on the mitochondrial membrane was successfully observed via rapid immunostaining without permeabilization.
[Conclusion]
Overall, the established cell unroofing method shows great potential to examine the localization, functions, and affinities of proteins on intact organelle membranes.
Molecular networks of organelle membranes are involved in many cell processes. However, the nature of plasma membrane as a barrier to various analytical tools, including antibodies, makes it challenging to examine intact organelle membranes without affecting their structure and functions via membrane permeabilization. For now, to overcome the problem some "cell unroofing" methods have been developed, in which cells are fractured with sonication probe and so on, and intracellular membrane surface is exposed. However, those methods examined only over intracellular surface of plasma membrane and but over organelle membrane surface, due to the loss of whole intracellular compartment under unroofing treatment. Therefore, in this study, we aimed to develop a microfluidic method to unroof cells and observe the intrinsic membrane molecules in organelles.
[Method]
In our method, single cells were precisely arrayed on the bottom surface of microchannels in a light-guided manner using a photoactivatable cell anchoring material. Then, induced fast laminar flax was hit on cells and physically some part of them was flowed away. However, this stress on cells can be controled by cell intervals. In detail, when cell intervals were long, flax hit onto the side of cells and most cell compartments without bottom plasma membrane were removed. On the other hand, at sufficiently short cell intervals, horizontal stresses generated by the laminar flow cannot approach the cell side and instantly fractured only the upper cell membranes, without significantly affecting some organelles inside the fractured cells. This was also supported by computational fluid dynamics simulation of flow around cells.
[Results and Consideration]
Subsequently, left compartment in unroofed cells were observed via confocal fluorescence and scanning electron microscopy. Confocal observation showed the loss of apical cell membrane and remained calcein-stained cytoplasmic compartment. Moreover, electron microscopy suggested the existence of some kinds of organelles including nucleus and mitochondria. Next, the healthiness of left organelle was confirmed through the detection of membrane potential, which is an indicator for damage on mitochondria. As a result, unroofed cells were suggested to sustain the membrane potential and to be protected from significant damage. Furthermore, distribution of the mitochondrial membrane protein, translocase of outer mitochondrial membrane 20, on the mitochondrial membrane was successfully observed via rapid immunostaining without permeabilization.
[Conclusion]
Overall, the established cell unroofing method shows great potential to examine the localization, functions, and affinities of proteins on intact organelle membranes.
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