Division of Nanophysiology

Introduction

Our group works on dynamic processes that take place at the plasma membrane. The plasma membrane surrounding each of our cells is not a rigid structure, but can be remodeled into different shapes with the help of complex protein machineries. This makes it possible to reconcile the essential barrier function of the membrane with the necessary selective uptake of surface proteins for the internalization of nutrients, the regulation of signal cascades and the remodeling of cellular adhesion complexes. A major uptake mechanism is endocytosis, a process in which the membrane is progressively bend inwards to form a vesicle which in the end is fissioned off to deliver its cargo to intracellular compartments.

 

Endocytosis allows the efficient uptake of nutrient, signaling and adhesion receptors and enables the regeneration of synaptic vesicles. Consequently, defects in this process can cause diseases ranging from anemia to epilepsy.

The extent to which endocytosis contributes to membrane dynamics can be appreciated from the fact that up to 50% of the cell surface is internalized by endocytosis per hour.
In this context, my research group is investigating questions such as:
- How does the cell ensure that the correct surface proteins are internalized?
- What are the consequences for the cell and the organism if the endocytosis of specific proteins is disturbed?

In particular, we try to answer questions such as:
- How do endocytic proteins impact brain functions such as our ability to forget?
- Which impact do endocytic proteins have on cell adhesion and migration?
- Can endocytosis help in the adaptation to cellular stress?

Endocytosis crucially impacts brain function

The brain is particularly sensitive to defects in endocytosis. Impairments in the uptake of individual synaptic proteins are already sufficient to cause serious neurological disorders. The functioning of the brain depends on the communication of neurons via neurotransmitters. The signal-sending neuron stores neurotransmitters in synaptic vesicles which fuse with the membrane upon a Ca2+ stimulus. The released neurotransmitters bind to neurotransmitter receptors on the neighbouring neuron thereby relaying the signal. Efficient neurotransmission relies on the local recycling of synaptic vesicles, which requires the efficient retrieval of transmembrane synaptic vesicle proteins from the neuronal membrane after vesicle fusion. Consequently, neurons depend on fast and reliable sorting processes to regenerate synaptic vesicles of the right protein composition for sustained neurotransmission.

In the past we have demonstrated that efficient neurotransmission relies on the use of dedicated endocytic adaptors to internalize crucial synaptic vesicle proteins. A prominent example is the importance of the endocytic adaptor AP180 for the retrieval of the vesicular SNARE protein Synaptobrevin2 which is crucial for synaptic vesicle fusion. Even though Synaptobrevin2 is a very abundant protein on synaptic vesicles, its partial missorting to the plasma membrane upon loss of AP180 has dramatic consequences for the organism. AP180 deficient mice suffer from reduced neurotransmission, seizures and premature death. Thus high-fidelity retrieval of synaptobrevin2 by AP180 is indeed crucial for brain function and survival (Koo et al., Neuron, 2015).

Currently, we are interested in the physiological role of the AP180 related protein CALM which has been linked to Alzheimer´s disease and also in the function of the endocytic adaptor proteins intersectin 1 and intersectin 2 whose loss impairs viability and leads to behavioral alterations such as repetitive jumping in mice.

 

 

Cargo-specific adaptors facilitate the sorting of synaptic vesicle (SV) proteins at the plasma membrane and on endosome-like vacuoles (ELVs). CME, clathrin-mediated endocytosis; CIE, clathrin-independent endocytosis [image modified from Kaempf & Maritzen, Safeguards of Neurotransmission: Endocytic Adaptors as Regulators of Synaptic Vesicle Composition and Function, Frontiers in Cellular Neuroscience, 2017].

Endocytosis helps cells to adapt to stress conditions

Cellular life is challenged by a multitude of stress conditions, triggered for example by alterations in osmolarity, oxygen or nutrient supply. The plasma membrane is not only the first point of encounter for many types of environmental stress, but given the diversity of receptor proteins and their associated molecules also represents the site at which many cellular signal cascades originate. Since these signaling pathways affect virtually all aspects of cellular life, changes in the plasma membrane proteome appear ideally suited to contribute to the cellular adaptation to stress. Since endocytosis can rapidly alter the surface proteome, it seems a likely contributer to the cellular stress response, however, very little is known so far about the actual role of endocytosis during cellular stress.

We could recently show that osmotic stress impairs the endocytosis of a specific ion transporter, the Na+/H+ exchanger NHE7. In a cascade of events (see scheme below) the ensuing surface accumulation of NHE7 triggers an increase in the degradative capacity of the cell thereby helping to counteract the protein aggregation caused by a hyperosmotic environment and thus promoting cell survival (López-Hernández et al., Nature Cell Biology, 2020). In the future, we would like to address in an unbiased way in how far and how endocytosis promotes cell survival in different stress conditions.

 

Under iso-osmotic conditions the Na+/H+ exchanger NHE7 is continuously endocytosed, thereby limiting its transport activity at the cell surface. Upon hyperosmotic conditions, endocytosis of NHE7 is downregulated resulting in elevated NHE7 surface levels. Increased NHE7 activity at the plasma membrane elevates Na+ influx, which via the Na+/Ca2+ exchanger NCX1 leads to increased intracellular Ca2+ levels and the Ca2+/Calcineurin-mediated dephosphorylation of the transcription factor TFEB to induce lysosomal and autophagy gene expression. The ensuing increased cellular degradative capacity is beneficial for counteracting protein aggregation caused by hyperosmotic conditions and therefore promotes cell survival [image from López-Hernández, Haucke & Maritzen, Endocytosis in the adaptation to cellular stress, Cell Stress, 2020]

Regulation of cellular adhesion

Cells use elaborate protein complexes to adhere to the extracellular matrix. The best understood adhesion complexes are the so-called focal adhesions (FAs) which prominently contain integrins and integrin binding proteins as linkers between extracellular matrix components and the intracellular cytoskeleton. FAs are continuously remodeled in line with cellular demands. Their dynamic nature is especially apparent when adherent cells have to migrate, e.g. during embryonic development, wound healing or immune defense. Mesenchymal cell motility relies on the continuous generation of new adhesions at the cell's leading edge and on their coordinated disassembly under the advancing cell body. Estimates for the number of FA proteins have been skyrocketing from 180 to 2412 proteins with the latest proteomic studies. This compositional complexity likely necessitates elaborate assembly and disassembly mechanisms.

We have initially become interested in FAs because endocytosis was suggested to play a role in FA disassembly, and we could show in the past that the endocytic adaptor protein Stonin1 indeed influences FAs (Feutlinske et al., Nature Communications, 2015). Currently, we are trying to unravel the mechanisms shaping FA dynamics with an unbiased genome-wide siRNA screen. In addition, we have lately become interested also in reticular adhesions, an adhesion type that is important during mitosis and long time culture and enriched in endocytic proteins.

 

Left: Mammalian cell stained by immunofluorescence with markers for focal and reticular adhesions, nucleus depicted in blue [image courtesy of Fabian Lukas].
Right: Proposed FA disassembly mechanisms. Microtubule based delivery of “relaxing factors” [1], extracellular proteolysis of extracellular matrix components [2], autophagy and intracellular proteolysis by calpain [3] and endocytosis [4] were all suggested to be involved in FA disassembly. However, it remains unclear how these processes would be spatiotemporally coordinated and whether this is the full picture [scheme courtesy of Lennart Hofmann].

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