Konrad E. Zinsmaier

The GTPase dMiro is required for axonal transport of mitochondria to drosophila synapses

We have identified EMS-induced mutations in Drosophila Miro (dMiro), an atypical mitochondrial GTPase that is orthologous to human Miro (hMiro). Mutant dmiro animals exhibit defects in locomotion and die prematurely. Mitochondria in dmiro mutant muscles and neurons are abnormally distributed. Instead of being transported into axons and dendrites, mitochondria accumulate in parallel rows in neuronal somata. Mutant neuromuscular junctions (NMJs) lack presynaptic mitochondria, but neurotransmitter release and acute Ca2+ buffering is only impaired during prolonged stimulation. Neuronal, but not muscular, expression of dMiro in dmiro mutants restored viability, transport of mitochondria to NMJs, the structure of synaptic boutons, the organization of presynaptic microtubules, and the size of postsynaptic muscles. In addition, gain of dMiro function causes an abnormal accumulation of mitochondria in distal synaptic boutons of NMJs. Together, our findings suggest that dMiro is required for controlling anterograde transport of mitochondria and their proper distribution within nerve terminals.

A Cysteine-String Protein is Expressed in Retina and Brain of Drosophila

Antibodies can be used to identify tissue- and stage-specifically expressed genes. A monoclonal antibody MAB ab49 from a hybridoma library screened for immunohistochemical staining in the adult nervous system of Drosophila melanogaster was found to selectively bind to all neuropil regions and to synaptic boutons of motor neurons. In Western blots of homogenized brains the antibody recognizes two proteins of 32 and 34 kD. Using this antibody we have isolated seven cDNA clones that derive from two polyadenylated mRNA splice variants of a gene located at 79E1-2 on polytene chromosomes. The two mRNAs code for two inferred proteins of 249 and 223 amino acids, respectively, which are identical except for their C-terminals and a central deletion of 21 amino acids in the second protein. Both contain a contiguous string of 11 cysteine residues. In situ hybridization to frozen head sections detects expression of this gene in retina and neuronal perikarya. The 32 and 34 kD brain proteins that presumably are localized predominantly in synaptic terminals of photoreceptors and most if not all neurons may correspond to two variant cysteine-string proteins as they are of similar molecular weight and share an antigenic binding site for MAB ab49.

Presynaptic Dysfunction in Drosophila csp Mutants

Cysteine string proteins are synapse-specific proteins. In Drosophila, csp deletion mutants exhibit temperature-sensitive paralysis and early death. Here, we report that neuromuscular transmission is impaired presynaptically in these csp mutant larvae. At 22 degrees C, evoked transmitter release is depressed relative to wild type and rescued controls, and high frequency stimulation of the nerve leads to sporadic failures. At 30 degrees C, stimulus-evoked responses decline gradually before failing completely. When the temperature is returned to 22 degrees C, evoked responses recover. Spontaneous release events persist at both 22 degrees C and 30 degrees C. Since nerve conduction and postsynaptic sensitivity are unaffected, these data indicate that csp mutations disrupt depolarization-secretion coupling. This disruption explains the cellular basis of the temperature-sensitive paralysis of these organisms.

Drosophila Miro is required for both anterograde and retrograde axonal mitochondrial transport

Microtubule-based transport of mitochondria into dendrites and axons is vital for sustaining neuronal function. Transport along microtubule tracks proceeds in a series of plus and minus end-directed movements that are facilitated by kinesin and dynein motors. How the opposing movements are controlled to achieve effective transport over large distances remains unclear. Previous studies showed that the conserved mitochondrial GTPase Miro is required for mitochondrial transport into axons and dendrites and serves as a Ca2+ sensor that controls mitochondrial mobility. To directly examine Miros significance for kinesin- and/or dynein-mediated mitochondrial motility, we live-imaged movements of GFP-tagged mitochondria in larval Drosophila motor axons upon genetic manipulations of Miro. Loss of Drosophila Miro (dMiro) reduced the effectiveness of both anterograde and retrograde mitochondrial transport by selectively impairing kinesin- or dynein-mediated movements, depending on the direction of net transport. Net anterogradely transported mitochondria exhibited reduced kinesin- but normal dynein-mediated movements. Net retrogradely transported mitochondria exhibited much shorter dynein-mediated movements, whereas kinesin-mediated movements were minimally affected. In both cases, the duration of short stationary phases increased proportionally. Overexpression (OE) of dMiro also impaired the effectiveness of mitochondrial transport. Finally, loss and OE of dMiro altered the length of mitochondria in axons through a mechanistically separate pathway. We suggest that dMiro promotes effective antero- and retrograde mitochondrial transport by extending the processivity of kinesin and dynein motors according to a mitochondrions programmed direction of transport.

Drosophila Hsc70-4 Is Critical for Neurotransmitter Exocytosis In Vivo

Previous in vitro studies of cysteine-string protein (CSP) imply a potential role for the clathrin-uncoating ATPase Hsc70 in exocytosis. We show that hypomorphic mutations in Drosophila Hsc70-4 (Hsc4) impair nerve-evoked neurotransmitter release, but not synaptic vesicle recycling in vivo. The loss of release can be restored by increasing external or internal Ca(2+) and is caused by a reduced Ca(2+) sensitivity of exocytosis downstream of Ca(2+) entry. Hsc4 and CSP are likely to act in common pathways, as indicated by their in vitro protein interaction, the similar loss of evoked release in individual and double mutants, and genetic interactions causing a loss of release in trans-heterozygous hsc4-csp double mutants. We suggest that Hsc4 and CSP cooperatively augment the probability of release by increasing the Ca(2+) sensitivity of vesicle fusion.

Membrane potential modulates plasma membrane phospholipid dynamics and K-Ras signaling

Membrane potential regulates growth Changes in electrical potential across the plasma membrane can affect cell growth. Zhou et al. discovered that membrane potential influenced the organization of phospholipids in the membrane of cultured mammalian cells and neurons in intact flies (see the Perspective by Accardi). This in turn regulated localization and activity of the small guanine nucleotide binding protein K-Ras, an important regulator of cell proliferation. The cell membrane may thus function analogously to a field-effect transistor by adjusting the strength of mitogenic signaling. Science, this issue p. 873; see also p. 789 Changing the voltage across the plasma membrane causes clustering of a small guanosine triphosphatase. [Also see Perspective by Accardi] Plasma membrane depolarization can trigger cell proliferation, but how membrane potential influences mitogenic signaling is uncertain. Here, we show that plasma membrane depolarization induces nanoscale reorganization of phosphatidylserine and phosphatidylinositol 4,5-bisphosphate but not other anionic phospholipids. K-Ras, which is targeted to the plasma membrane by electrostatic interactions with phosphatidylserine, in turn undergoes enhanced nanoclustering. Depolarization-induced changes in phosphatidylserine and K-Ras plasma membrane organization occur in fibroblasts, excitable neuroblastoma cells, and Drosophila neurons in vivo and robustly amplify K-Ras–dependent mitogen-activated protein kinase (MAPK) signaling. Conversely, plasma membrane repolarization disrupts K-Ras nanoclustering and inhibits MAPK signaling. By responding to voltage-induced changes in phosphatidylserine spatiotemporal dynamics, K-Ras nanoclusters set up the plasma membrane as a biological field-effect transistor, allowing membrane potential to control the gain in mitogenic signaling circuits.

Molecular chaperones and the regulation of neurotransmitter exocytosis.

Regulated neurotransmitter release depends on a precise sequence of events that lead to repeated cycles of exocytosis and endocytosis. These events are mediated by a series of molecular interactions among vesicular, plasma membrane, and cytosolic proteins. An emerging theme has been that molecular chaperones may guide the sequential restructuring of stable or transient protein complexes to promote a temporal and spatial regulation of the endo- and exocytotic machinery and to ensure a vectorial passage through the vesicle cycle. Chaperones, specialized for a few substrates, are ideally suited to participate in regulatory processes that require some molecular dexterity to rearrange conformational or oligomeric protein structures. This article emphasizes the significance of three molecular chaperone systems in regulated neurotransmitter release: the regulation of soluble NSF attachment protein receptor (SNARE) complexes by N-ethylmaleimide-sensitive factor (NSF) and the soluble NSF attachment protein (SNAP), the uncoating of clathrin-coated vesicles by the 70 kDa heat-shock cognate protein (Hsc70), and the regulation of SNARE complex-associated protein interactions by cysteine-string protein and Hsc70.

Presynaptic Regulation of Neurotransmission in Drosophila by the G Protein-Coupled Receptor Methuselah

Regulation of synaptic strength is essential for neuronal information processing, but the molecular mechanisms that control changes in neuroexocytosis are only partially known. Here we show that the putative G protein-coupled receptor Methuselah (Mth) is required in the presynaptic motor neuron to acutely upregulate neurotransmitter exocytosis at larval Drosophila NMJs. Mutations in the mth gene reduce evoked neurotransmitter release by approximately 50%, and decrease synaptic area and the density of docked and clustered vesicles. Pre- but not postsynaptic expression of normal Mth restored normal release in mth mutants. Conditional expression of Mth restored normal release and normal vesicle docking and clustering but not the reduced size of synaptic sites, suggesting that Mth acutely adjusts vesicle trafficking to synaptic sites.

The Multiple Functions of Cysteine-String Protein Analyzed at Drosophila Nerve Terminals

The synaptic vesicle-associated cysteine-string protein (CSP) is important for synaptic transmission. Previous studies revealed multiple defects at neuromuscular junctions (NMJs) of csp null-mutant Drosophila, but whether these defects are independent of each other or mechanistically linked through J domain mediated-interactions with heat-shock cognate protein 70 (Hsc70) has not been established. To resolve this issue, we genetically dissected the individual functions of CSP by an in vivo structure/function analysis. Expression of mutant CSP lacking the J domain at csp null-mutant NMJs fully restored normal thermo-tolerance of evoked transmitter release but did not completely restore evoked release at room temperature and failed to reverse the abnormal intraterminal Ca2+ levels. This suggests that J domain-mediated functions are essential for the regulation of intraterminal Ca2+ levels but only partially required for regulating evoked release and not required for protecting evoked release against thermal stress. Hence, CSP can also act as an Hsc70-independent chaperone protecting evoked release from thermal stress. Expression of mutant CSP lacking the L domain restored neurotransmission and partially reversed the abnormal intraterminal Ca2+ levels, suggesting that the L domain is important, although not essential, for the role of CSP in regulating intraterminal Ca2+ levels. We detected no effects of csp mutations on individual presynaptic Ca2+ signals triggered by action potentials, suggesting that presynaptic Ca2+ entry is not primarily impaired. Both the J and L domains were also required for the role of CSP in synaptic growth. Together, these results suggest that CSP has several independent synaptic functions, affecting synaptic growth, evoked release, thermal protection of evoked release, and intraterminal Ca2+ levels at rest and during stimulation.

Presynaptic Mitochondria in Functionally Different Motor Neurons Exhibit Similar Affinities for Ca2+ But Exert Little Influence as Ca2+ Buffers at Nerve Firing Rates In Situ

Mitochondria accumulate within nerve terminals and support synaptic function, most notably through ATP production. They can also sequester Ca2+ during nerve stimulation, but it is unknown whether this limits presynaptic Ca2+ levels at physiological nerve firing rates. Similarly, it is unclear whether mitochondrial Ca2+ sequestration differs between functionally different nerve terminals. We addressed these questions using a combination of synthetic and genetically encoded Ca2+ indicators to examine cytosolic and mitochondrial Ca2+ levels in presynaptic terminals of tonic (MN13-Ib) and phasic (MNSNb/d-Is) motor neurons in Drosophila, which, as we determined, fire during fictive locomotion at ∼42 Hz and ∼8 Hz, respectively. Mitochondrial Ca2+ sequestration starts in both terminals at ∼250 nm, exhibits a similar Ca2+-uptake affinity (∼410 nm), and does not require Ca2+ release from the endoplasmic reticulum. Nonetheless, mitochondrial Ca2+ uptake in type Is terminals is more responsive to low-frequency nerve stimulation and this is due to higher cytosolic Ca2+ levels. Since type Ib terminals have a higher mitochondrial density than Is terminals, it seemed possible that greater mitochondrial Ca2+ sequestration may be responsible for the lower cytosolic Ca2+ levels in Ib terminals. However, genetic and pharmacological manipulations of mitochondrial Ca2+ uptake did not significantly alter nerve-stimulated elevations in cytosolic Ca2+ levels in either terminal type within physiologically relevant rates of stimulation. Our findings indicate that presynaptic mitochondria have a similar affinity for Ca2+ in functionally different nerve terminals, but do not limit cytosolic Ca2+ levels within the range of motor neuron firing rates in situ.