Hans-Hermann Gerdes

Green fluorescent protein: applications in cell biology

The green fluorescent protein (GFP) of Aequorea victoria is a unique in vivo reporter for monitoring dynamic processes in cells or organisms. As a fusion tag GFP can be used to localize proteins, to follow their movement or to study the dynamics of the subcellular compartments to which these proteins are targeted. Recent studies where GFP technology has revealed new insights regarding physiological activities of living cells are discussed.

Intercellular transfer mediated by tunneling nanotubes.

Animal cells have evolved different mechanisms to communicate with one another. In 2004, a new route of cell-to-cell communication mediated by tunneling nanotubes (TNT) was reported. These membranous cell bridges form de novo between cells and mediate the intercellular transfer of organelles, plasma membrane components and cytoplasmic molecules. The characterization of TNT-like bridges from several cell types revealed variations in the cytoskeletal composition as well as in the modality by which they interconnect cells, suggesting that different subclasses may exist. Furthermore, the growing number of cell types for which TNT-like structures were detected, supports the view that they represent a general mechanism for functional connectivity between cells, which could have important implications under physiological conditions.

Tunneling nanotubes: A new route for the exchange of components between animal cells

Recently, highly sensitive nanotubular structures mediating membrane continuity between mammalian cells have been discovered. With respect to their peculiar architecture, these membrane channels were termed tunneling nanotubes (TNTs). TNTs could form de novo between animal cells leading to the generation of complex cellular networks. They have been shown to facilitate the intercellular transfer of organelles as well as, on a limited scale, of membrane components and cytoplasmic molecules. It has been proposed that TNTs represent a novel and general biological principle of cell‐to‐cell communication and it becomes increasingly apparent that they fulfill important functions in the physiological processes of multicellular organisms.

Visualization of protein transport along the secretory pathway using green fluorescent protein

We have expressed green fluorescent protein (GFP) from A. victoria in the secretory pathway of HeLa cells by fusing it to the C‐terminus of a secretory protein, chromogranin B. Under normal culture conditions at 37°C maturation of GFP to the fluorescent form was not detectable. However, fluorescent GFP was observed when biosynthetic protein transport was arrested at the intermediate compartment or the trans‐Golgi network by temperature blocks (15°C and 20°C, respectively). Reversal of the temperature blocks allowed the visualization of secretion of fluorescent GFP and offers the possibility to analyse transport in the secretory pathway in living cells.

Animal cells connected by nanotubes can be electrically coupled through interposed gap-junction channels

Tunneling nanotubes (TNTs) are recently discovered conduits for a previously unrecognized form of cell-to-cell communication. These nanoscale, F-actin–containing membrane tubes connect cells over long distances and facilitate the intercellular exchange of small molecules and organelles. Using optical membrane-potential measurements combined with mechanical stimulation and whole-cell patch-clamp recording, we demonstrate that TNTs mediate the bidirectional spread of electrical signals between TNT-connected normal rat kidney cells over distances of 10 to 70 μm. Similar results were obtained for other cell types, suggesting that electrical coupling via TNTs may be a widespread characteristic of animal cells. Strength of electrical coupling depended on the length and number of TNT connections. Several lines of evidence implicate a role for gap junctions in this long-distance electrical coupling: punctate connexin 43 immunoreactivity was frequently detected at one end of TNTs, and electrical coupling was voltage-sensitive and inhibited by meclofenamic acid, a gap-junction blocker. Cell types lacking gap junctions did not show TNT-dependent electrical coupling, which suggests that TNT-mediated electrical signals are transmitted through gap junctions at a membrane interface between the TNT and one cell of the connected pair. Measurements of the fluorescent calcium indicator X-rhod-1 revealed that TNT-mediated depolarization elicited threshold-dependent, transient calcium signals in HEK293 cells. These signals were inhibited by the voltage-gated Ca2+ channel blocker mibefradil, suggesting they were generated via influx of calcium through low voltage-gated Ca2+ channels. Taken together, our data suggest a unique role for TNTs, whereby electrical synchronization between distant cells leads to activation of downstream target signaling.

Myosin Va facilitates the distribution of secretory granules in the F-actin rich cortex of PC12 cells

Neuroendocrine secretory granules, the storage organelles for neuropeptides and hormones, are formed at the trans-Golgi network, stored inside the cell and exocytosed upon stimulation. Previously, we have reported that newly formed secretory granules of PC12 cells are transported in a microtubule-dependent manner from the trans-Golgi network to the F-actin-rich cell cortex, where they undergo short directed movements and exhibit a homogeneous distribution. Here we provide morphological and biochemical evidence that myosin Va is associated with secretory granules. Expression of a dominant-negative tail domain of myosin Va in PC12 cells led to an extensive clustering of secretory granules close to the cell periphery, a loss of their cortical restriction and a strong reduction in their motility in the actin cortex. Based on this data we propose a model that implies a dual transport system for secretory granules: after microtubule-dependent delivery to the cell periphery, secretory granules exhibit a myosin Va-dependent transport leading to their restriction and even dispersal in the F-actin-rich cortex of PC12 cells.

The disulfide‐bonded loop of chromogranin B mediates membrane binding and directs sorting from the trans‐Golgi network to secretory granules

The disulfide‐bonded loop of chromogranin B (CgB), a regulated secretory protein with widespread distribution in neuroendocrine cells, is known to be essential for the sorting of CgB from the trans‐Golgi network (TGN) to immature secretory granules. Here we show that this loop, when fused to the constitutively secreted protein α1‐antitrypsin (AT), is sufficient to direct the fusion protein to secretory granules. Importantly, the sorting efficiency of the AT reporter protein bearing two loops (E2/3–AT–E2/3) is much higher compared with that of AT with a single disulfide‐bonded loop. In contrast to endogenous CgB, E2/3–AT–E2/3 does not undergo Ca2+/pH‐dependent aggregation in the TGN. Furthermore, the disulfide‐bonded loop of CgB mediates membrane binding in the TGN and does so with 5‐fold higher efficiency if two loops are present on the reporter protein. The latter finding supports the concept that under physiological conditions, aggregates of CgB are the sorted units of cargo which have multiple loops on their surface leading to high membrane binding and sorting efficiency of CgB in the TGN.

Protein secretion: Puzzling receptors

All known sorting receptors for soluble cargo in the secretory pathway are transmembrane proteins. For sorting to the regulated pathway, however, a subpopulation of secretory proteins, associated with the membrane but not membrane-spanning, appears to link cargo and membrane in storage granule biogenesis.

Transfer of mitochondria via tunneling nanotubes rescues apoptotic PC12 cells

Tunneling nanotubes (TNTs) are F-actin-based membrane tubes that form between cells in culture and in tissues. They mediate intercellular communication ranging from electrical signalling to the transfer of organelles. Here, we studied the role of TNTs in the interaction between apoptotic and healthy cells. We found that pheochromocytoma (PC) 12 cells treated with ultraviolet light (UV) were rescued when cocultured with untreated PC12 cells. UV-treated cells formed a different type of TNT with untreated PC12 cells, which was characterized by continuous microtubule localized inside these TNTs. The dynamic behaviour of mCherry-tagged end-binding protein 3 and the accumulation of detyrosinated tubulin in these TNTs indicate that they are regulated structures. In addition, these TNTs show different biophysical properties, for example, increased diameter allowing dye entry, prolonged lifetime and decreased membrane fluidity. Further studies demonstrated that microtubule-containing TNTs were formed by stressed cells, which had lost cytochrome c but did not enter into the execution phase of apoptosis characterized by caspase-3 activation. Moreover, mitochondria colocalized with microtubules in TNTs and transited along these structures from healthy to stressed cells. Importantly, impaired formation of TNTs and untreated cells carrying defective mitochondria were unable to rescue UV-treated cells in the coculture. We conclude that TNT-mediated transfer of functional mitochondria reverse stressed cells in the early stages of apoptosis. This provides new insights into the survival mechanisms of damaged cells in a multicellular context.

Selective block of tunneling nanotube (TNT) formation inhibits intercellular organelle transfer between PC12 cells

Organelle exchange between cells via tunneling nanotubes (TNTs) is a recently described form of intercellular communication. Here, we show that the selective elimination of filopodia from PC12 cells by 350 nM cytochalasin B (CytoB) blocks TNT formation but has only a weak effect on the stability of existing TNTs. Under these conditions the intercellular organelle transfer was strongly reduced, whereas endocytosis and phagocytosis were not affected. Furthermore, the transfer of organelles significantly correlated with the presence of a TNT‐bridge. Thus, our data support that in PC12 cells filopodia‐like protrusions are the principal precursors of TNTs and CytoB provides a valuable tool to selectively interfere with TNT‐mediated cell‐to‐cell communication.