Gertrude Bunt

Neuron to glia signaling triggers myelin membrane exocytosis from endosomal storage sites

During vertebrate brain development, axons are enwrapped by myelin, an insulating membrane produced by oligodendrocytes. Neuron-derived signaling molecules are temporally and spatially required to coordinate oligodendrocyte differentiation. In this study, we show that neurons regulate myelin membrane trafficking in oligodendrocytes. In the absence of neurons, the major myelin membrane protein, the proteolipid protein (PLP), is internalized and stored in late endosomes/lysosomes (LEs/Ls) by a cholesterol-dependent and clathrin-independent endocytosis pathway that requires actin and the RhoA guanosine triphosphatase. Upon maturation, the rate of endocytosis is reduced, and a cAMP-dependent neuronal signal triggers the transport of PLP from LEs/Ls to the plasma membrane. These findings reveal a fundamental and novel role of LEs/Ls in oligodendrocytes: to store and release PLP in a regulated fashion. The release of myelin membrane from LEs/Ls by neuronal signals may represent a mechanism to control myelin membrane growth.

Myelin basic protein-dependent plasma membrane reorganization in the formation of myelin

During vertebrate development, oligodendrocytes wrap their plasma membrane around axons to produce myelin, a specialized membrane highly enriched in galactosylceramide (GalC) and cholesterol. Here, we studied the formation of myelin membrane sheets in a neuron–glia co‐culture system. We applied different microscopy techniques to visualize lipid packing and dynamics in the oligodendroglial plasma membrane. We used the fluorescent dye Laurdan to examine the lipid order with two‐photon microscopy and observed that neurons induce a dramatic lipid condensation of the oligodendroglial membrane. On a nanoscale resolution, using stimulated emission depletion and fluorescence resonance energy transfer microscopy, we demonstrated a neuronal‐dependent clustering of GalC in oligodendrocytes. Most importantly these changes in lipid organization of the oligodendroglial plasma membrane were not observed in shiverer mice that do not express the myelin basic protein. Our data demonstrate that neurons induce the condensation of the myelin‐forming bilayer in oligodendrocytes and that MBP is involved in this process of plasma membrane rearrangement. We propose that this mechanism is essential for myelin to perform its insulating function during nerve conduction.

Visualization of molecular activities inside living cells with fluorescent labels

The major task of modern cell biology is to identify the function and relation of the many different gene products, discovered by genomics and proteomics approaches, in the context of the living cell. To achieve this goal, an increasing toolbox of custom-designed biosensors based on fluorescent labels is available to study the molecular activities of the cellular machinery. An overview of the current status of the young field of molecular-cellular physiology is presented that includes the application of fluorescent labels in the design of biosensors and the major detection schemes used to extract their sensing information. In particular, the use of the photophysical phenomenon of Förster resonance energy transfer (FRET) as a powerful indicator of cellular biochemical events is discussed. In addition, we will point out the challenges and directions of the field and project the short-term future for the application of fluorescence-based biosensors in biology.

Resolution doubling in fluorescence microscopy with confocal spinning-disk image scanning microscopy

Significance Fluorescence microscopy is an enormously powerful tool for investigating structural organization and dynamical processes on the cellular level because of its noninvasiveness, sensitivity, and specificity. However, classical fluorescence microscopy is limited in resolution by the diffraction of light. In recent years, structural illumination microscopy has succeeded in doubling this resolution without requiring any special sample preparation or labeling dyes. However, it is technically very challenging and complex. Here, we present an alternative that achieves the same resolution enhancement by using a standard confocal spinning-disk microscope with minimal modifications. This method, in principle, allows one to double the resolution of any existing confocal microscope. We demonstrate how a conventional confocal spinning-disk (CSD) microscope can be converted into a doubly resolving image scanning microscopy (ISM) system without changing any part of its optical or mechanical elements. Making use of the intrinsic properties of a CSD microscope, we illuminate stroboscopically, generating an array of excitation foci that are moved across the sample by varying the phase between stroboscopic excitation and rotation of the spinning disk. ISM then generates an image with nearly doubled resolution. Using conventional fluorophores, we have imaged single nuclear pore complexes in the nuclear membrane and aggregates of GFP-conjugated Tau protein in three dimensions. Multicolor ISM was shown on cytoskeletal-associated structural proteins and on 3D four-color images including MitoTracker and Hoechst staining. The simple adaptation of conventional CSD equipment allows superresolution investigations of a broad variety of cell biological questions.

Dynamic conformational changes in the FERM domain of FAK are involved in focal-adhesion behavior during cell spreading and motility.

Focal adhesion kinase (FAK) controls cellular adhesion and motility processes by its tight link to integrin- and extracellular-matrix-mediated signaling. To explore the dynamics of the regulation of FAK, we constructed a FRET-based probe that visualizes conformational rearrangements of the FERM domain of FAK in living cells. The sensor reports on an integrin-mediated conformational change in FAK following cellular adhesion. The perturbation is kinase-independent and involves the polybasic KAKTLR sequence in the FERM domain. It is manifested by an increased FRET signal and is expressed primarily in focal adhesions, and to a lesser extent in the cytoplasm. The conformational change in the FERM domain of FAK is observed in two consecutive phases during spreading – early and late – and is enriched in fully adhered motile cells at growing and sliding peripheral focal-adhesion sites, but not in stable or retracting focal adhesions. Inhibition of the actomyosin system indicates the involvement of tension signaling induced by Rho-associated kinase, rather than by myosin light-chain kinase, in the modulation of the FERM response. We conclude that the heterogeneous conformation of the FERM domain in focal adhesions of migrating cells reflects a complex regulatory mechanism for FAK that appears to be under the influence of cellular traction forces.

Removal of pattern-breaking sequences in microtubule binding repeats produces instantaneous tau aggregation and toxicity.

Aggregated and highly phosphorylated tau protein is a pathological hallmark of Alzheimers disease (AD) and other tauopathies. We identified motifs of alternating polar and apolar amino acids within the microtubule-binding repeats of tau which were interrupted by small breaking stretches. Minimal mutation of these breaking sequences yielded a unique instantly aggregating tau mutant containing longer stretches of polar/apolar amino acids without losing its microtubule-binding capacity. These modifications produced rapid aggregation and cytotoxicity with accompanying occurrence of pathologic tau phosphoepitopes (AT8, AT180, AT270, AT100, Ser422, and PHF-1) and conformational epitopes (MC-1 and Alz50) in cells. Similar to pathological tau in the pretangle state, toxicity appeared to occur early without the requirement for extensive fibril formation. Thus, our mutant protein provides a novel platform for the investigation of the molecular mechanisms for toxicity and cellular behavior of pathologically aggregated tau proteins and the identification of its interaction partners.

Dynamic Conformational Transitions of the EGF Receptor in Living Mammalian Cells Determined by FRET and Fluorescence Lifetime Imaging Microscopy

We have revealed a reorientation of ectodomain I of the epidermal growth factor receptor (EGFR; ErbB1; Her1) in living CHO cells expressing the receptor, upon binding of the native ligand EGF. The state of the unliganded, nonactivated EGFR was compared to that exhibited after ligand addition in the presence of a kinase inhibitor that prevents endocytosis but does not interfere with binding or the ensuing conformational rearrangements. To perform these experiments, we constructed a transgene EGFR with an acyl carrier protein sequence between the signal peptide and the EGFR mature protein sequence. This protein, which behaves similarly to wild‐type EGFR with respect to EGF binding, activation, and internalization, can be labeled at a specific serine in the acyl carrier tag with a fluorophore incorporated into a 4′‐phosphopantetheine (P‐pant) conjugate transferred enzymatically from the corresponding CoA derivative. By measuring Förster resonance energy transfer between a molecule of Atto390 covalently attached to EGFR in this manner and a novel lipid probe NR12S distributed exclusively in the outer leaflet of the plasma membrane, we determined the apparent relative separation of ectodomain I from the membrane under nonactivating and activating conditions. The data indicate that the unliganded domain I of the EGFR receptor is situated much closer to the membrane before EGF addition, supporting the model of a self‐inhibited configuration of the inactive receptor in quiescent cells.