Abstract
Microtubules are a vital component of the cell’s cytoskeleton and their organization is crucial for healthy cell functioning. The use of label-free SH imaging of microtubules remains limited, as sensitive detection is required and the true molecular origin and main determinants required to generate SH from microtubules are not fully understood. Using advanced correlative imaging techniques, we identified the determinants of the microtubule-dependent SH signal. Microtubule polarity, number and organization determine SH signal intensity in biological samples. At the molecular level, we show that the GTP-bound tubulin dimer conformation is fundamental for microtubules to generate detectable SH signals. We show that SH imaging can be used to study the effects of microtubule-targeting drugs and proteins and to detect changes in tubulin conformations during neuronal maturation. Our data provide a means to interpret and use SH imaging to monitor changes in the microtubule network in a label-free manner.
Highlights
Microtubules are a vital component of the cell’s cytoskeleton and their organization is crucial for healthy cell functioning
Combining second harmonic (SH) imaging with correlative fluorescence and electron microscopy (CLEM), we demonstrate the importance of theoretical determinants such as microtubule orientation, number, and organization in biological samples where multiple microtubules are present
To validate the microtubular origin of the detected SH signals, we show that in vitro polymerized microtubules lacking other cellular components are capable of generating SH (Fig. 1c) and that in living cells expressing eGFP-labeled microtubules the fluorescence colocalizes with the SH signals (Fig. 1d)
Summary
Microtubules are a vital component of the cell’s cytoskeleton and their organization is crucial for healthy cell functioning. SHG is mainly used to visualize strong biological harmonophores like collagen[19,20,21], it can be used to detect the far weaker tubulin dimer as it organizes itself in the microtubule lattice[19,22,23,24,25,26] Though this technique does not require staining or fluorescence-based methods, possibly interfering with cellular processes, the use of SHG microscopy of microtubules is not yet established in neuroscience as the true molecular origin remains elusive and interpretation of the relatively weak signal is not trivial. We show how SHG imaging of microtubules can be used to study a variety of biological mechanisms in neurodegeneration and neuronal maturation
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