Biology subjects

Rizzoli, S. O.

Publications and source records attributed to Rizzoli, S. O..

23 records · Page 2Linked to original sources

bioRxiv2019

A nanobody-based fluorescent reporter reveals human α-synuclein in the cell cytosol

Aggregation and spreading of -Synuclein (Syn) are hallmarks of several neurodegenerative diseases, thus monitoring human Syn (hSyn) in animal models or cell cultures is vital for the field. However, the detection of native hSyn in such systems is challenging. We found that the nanobody NbSyn87, previously-described to bind hSyn, also shows cross-reactivity for the proteasomal subunit Rpn10. As such, when the NbSyn87 is expressed in the absence of hSyn, it is continuously degraded by the proteasome, while it is stabilized when it binds to hSyn. Here, we exploited this feature to design a new Fluorescent Reporter for hSyn (FluoReSyn) by coupling NbSyn87 to fluorescent proteins, which results in fluorescence signal fluctuations depending on the presence and amounts of intracellular hSyn. We characterized this biosensor in cells and tissues, and finally, revealed the presence of transmittable Syn in human cerebrospinal fluid demonstrating the potential of FluoReSyn for clinical research and diagnostics.

neuroscience↗

bioRxiv2019

Circumvention of common labeling artifacts using secondary nanobodies

The most common procedure to reveal the location of specific (sub)cellular elements in biological samples is via immunostaining followed by optical imaging. This is typically performed with target-specific primary antibodies (1.Abs), which are revealed by fluorophore-conjugated secondary antibodies (2.Abs). However, at high resolution this methodology can induce a series of artifacts due to the large size of antibodies, their bivalency, and their polyclonality. Here we use STED and DNA-PAINT super-resolution microscopy or light sheet microscopy on cleared tissue to show how monovalent secondary reagents based on camelid single-domain antibodies (nanobodies; 2.Nbs) attenuate these artifacts. We demonstrate that monovalent 2.Nbs have four additional advantages: 1) they increase localization accuracy with respect to 2.Abs; 2) they allow direct pre-mixing with 1.Abs before staining, reducing experimental time, and enabling the use of multiple 1.Abs from the same species; 3) they penetrate thick tissues efficiently; and 4) they avoid the artificial clustering seen with 2.Abs both in live and in poorly fixed samples. Altogether, this suggests that 2.Nbs are a valuable alternative to 2.Abs, especially when super-resolution imaging or staining of thick tissue samples are involved.

cell biology↗

bioRxiv2018

Nanobodies reveal an extra-synaptic population of SNAP-25 and Syntaxin 1A in hippocampal neurons

Synaptic vesicle fusion (exocytosis) is a precisely regulated process that entails the formation of SNARE complexes between the vesicle protein synaptobrevin 2 (VAMP2) and the plasma membrane proteins Syntaxin 1 and SNAP-25. The sub-cellular localization of the latter two molecules remains unclear, although they have been the subject of many recent investigations. To address this, we generated two novel camelid single domain antibodies (nanobodies) specifically binding to SNAP-25 and Syntaxin 1A. These probes penetrated more easily into samples and detected their targets more efficiently than conventional antibodies in crowded regions. When investigated by super-resolution imaging, the nanobodies revealed substantial extra-synaptic populations for both SNAP-25 and Syntaxin 1A, which were poorly detected by antibodies. Moreover, extra-synaptic Syntaxin 1A molecules were recruited to synapses during stimulation, suggesting that these are physiologically-active molecules. We conclude that nanobodies are able to reveal qualitatively and quantitatively different organization patterns, when compared to conventional antibodies.

neuroscience↗

bioRxiv2017

X10 Expansion Microscopy Enables 25 nm Resolution on Conventional Microscopes

Expansion microscopy is a recently introduced imaging technique that achieves super-resolution through physically expanding the specimen by ~4x, after embedding into a swellable gel. The resolution attained is, correspondingly, approximately 4-fold better than the diffraction limit, or ~70 nm. This is a major improvement over conventional microscopy, but still lags behind modern STED or STORM setups, whose resolution can reach 20-30 nm. We addressed this issue here by introducing an improved gel recipe that enables an expansion factor of ~10x in each dimension, which corresponds to an expansion of the sample volume by more than 1000-fold. Our protocol, which we termed X10 microscopy, achieves a resolution of 25-30 nm on conventional epifluorescence microscopes. X10 provides multi-color images similar or even superior to those produced with more challenging methods, such as STED, STORM and iterative expansion microscopy (iExM), in both cell cultures and tissues.

cell biology↗

bioRxiv2017

Ageing synaptic vesicles are inactivated by contamination with SNAP25

Old organelles can become a hazard to cellular function, by accumulating molecular damage. Mechanisms that identify aged organelles, and prevent them from participating in cellular reactions, are therefore necessary. We describe here one such mechanism for the synaptic vesicle recycling pathway. Using cultured hippocampal neurons, we found that newly synthesized vesicle proteins were incorporated in the active (recycling) pool, and were preferentially employed in neurotransmitter release. They remained in use for up to ~24 hours, during which they recycled up to a few hundred times. We could only detect one change in the molecular composition of the vesicles, an apparent accumulation of SNAP25 in the aged synaptic vesicles. Overexpression of SNAP25, both in wild-type form or in vesicle-bound form, inhibited exocytosis and promoted the co-localization of the vesicle molecules with a recycling endosome marker. This is in line with the hypothesis that the SNAP25 contamination causes the inactivation of the aged vesicles. The SNAP25 overexpression effect could be alleviated by co-expressing the vesicle-associated molecule CSP, which has been previously shown to be involved in chaperoning SNAP25 in the vesicle priming process. Overall, these results suggest that newly synthesized vesicle molecules are preferred in vesicle recycling, probably through a mechanism that renders their priming more efficient than that of aged vesicles.

neuroscience↗