Research

Neuronal synapses form the basis for neuronal communication and the storage of information in brain. The strength and persistence of chemical synapses are tightly regulated and the plastic properties of neighbored dendritic synapses are also determined by molecular and electrical signaling in dendritic segments. Potentiation of a dendritic spine favors the potentiation of its neighbor. In this regard, the dendritic branch forms a perfect compartment for confined signaling. The overall aim of our lab is to understand what defines a dendritic segment as a "plasticity unit" and what are the underlying molecular mechanisms of heterosynaptic plasticity. We take a multidisciplinary approach by combining electrophysiology with advanced imaging techniques and biochemical/molecular biological methods to tackle two main questions: 1. How are synaptic proteins involved in interactions between nearby synapses and how do they contribute to the establishment of clustered synaptic plasticity within a dendritic branch? 2. What is the role of dendritic secretory trafficking organelles like ER-Golgi intermediate compartment (ERGIC), the dendritic Golgi satellite system, retromer and endosomal systems in establishment and maintenance of dendritic compartments? In this respect understanding how the interplay between motor proteins allows for controlled cargo delivery, retention or release in response to synaptic activity in dendritic branch compartments might be of key importance. Our studies are currently focused on 3 main projects:

1. Neuronal calcium binding proteins in spine function

Ca2+ signaling plays an essential role in neuronal signal transmission and synaptic plasticity. Intracellular Ca2+ levels are tightly regulated and cells undertakes enormous work to keep basal Ca2+ levels low and to enable Ca2+ rise upon signaling, e.g. neurotransmitter release at the presynapse upon arrival of an action potential and postsynaptic activation of signaling cascade upon neurotransmitter binding to receptors and channel opening.
There is a plethora of Ca2+ binding proteins that form the basis for the spatial and temporal transduction of Ca2+ transients. Ca2+ sensor proteins provide high signaling specificity by triggering target interactions. We investigate the neuronal Ca2+ binding protein caldendrin, which is highly enriched in dendritic spines and its function in spine dynamics. In vitro characterization of novel interactions of caldendrin is combined with a knockout mice approach to study the physiological role of caldendrin. Currently a major methodological focus in this project lies on primary cell culture and organotypic slice culture combined with electrophysiology and confocal microscopy.

Mikhaylova

Organotypic hippocampal slice biolistically transfected with GFP. DAPI (cyan), GFP (yellow)
Mikhaylova

Organotypic hippocampal slice biolistically transfected with GFP. DAPI (cyan), GFP (yellow)

2. Molecular mechanisms of heterosynaptic plasticity

Neighboring synapses cooperate and influence each other's fate. LTP at a single synapse promotes the induction of LTP in adjacent synapses. The proposed underlying signaling involves activation and diffusion of signaling molecules, and the local synthesis of new plasticity-related proteins. We hypothesize that redistribution of synaptic scaffolding and signaling proteins can be an important factor defining boundaries of dendritic compartments containing clustered spines. The aim of this project is to learn more about sharing of synaptic proteins by means of diffusion and active transport and the role of dendritic secretory organelles in dendritic compartmentalization. Specifically we ask what is the turnover rate of proteins in active vs. inactive spines? How does synaptic turnover correlate with spine size and morphology? Do these proteins exit and move as a cluster? How far do they travel? Does the protein remain within dendritic branch points? And does secretory trafficking through dendritic ER and Golgi satellite organelles play a role in the maintenance of dendritic segments? We tackle these questions by using novel tools namely: nanobody/intrabody labeling and light-controlled transport assays. This is combined with established techniques such as glutamate uncaging, electrophysiology, confocal and 2-photon microscopy.

Mikhaylova

Super resolution microscope
Mikhaylova

Two-photon microscope

3. Nanobodies/intrabodies for studying the localization and dynamics of postsynaptic proteins

Many live-cell imaging studies suggest that different types of synaptic proteins (receptors, synaptic cell adhesion molecules, scaffolding and signaling proteins) continuously move in, out and between synapses. We would like to gain a better understanding of the dynamics of endogenous synaptic proteins upon induction of heterosynaptic plasticity. Therefore we are putting major efforts in developing novel molecular tools that allow improved visualization, tracing and the controlled redistribution of endogenous mobile candidate proteins. We are currently generating nanobody (also called VHHs, variable domain of the heavy chain of a heavy chain-only antibody from cameloids) and intrabody (based on natural scaffolds like the 10th fibronectin type III domain of human fibronectin) libraries, which will be used for selection of binders for chosen synaptic targets.

Mikhaylova

'Our' alpacas at the Preclinics GmbH farm

Cooperations