Dale W. Hailey

Mitochondria Supply Membranes for Autophagosome Biogenesis during Starvation

Starvation-induced autophagosomes engulf cytosol and/or organelles and deliver them to lysosomes for degradation, thereby resupplying depleted nutrients. Despite advances in understanding the molecular basis of this process, the membrane origin of autophagosomes remains unclear. Here, we demonstrate that, in starved cells, the outer membrane of mitochondria participates in autophagosome biogenesis. The early autophagosomal marker, Atg5, transiently localizes to punctae on mitochondria, followed by the late autophagosomal marker, LC3. The tail-anchor of an outer mitochondrial membrane protein also labels autophagosomes and is sufficient to deliver another outer mitochondrial membrane protein, Fis1, to autophagosomes. The fluorescent lipid NBD-PS (converted to NBD-phosphotidylethanolamine in mitochondria) transfers from mitochondria to autophagosomes. Photobleaching reveals membranes of mitochondria and autophagosomes are transiently shared. Disruption of mitochondria/ER connections by mitofusin2 depletion dramatically impairs starvation-induced autophagy. Mitochondria thus play a central role in starvation-induced autophagy, contributing membrane to autophagosomes.

Ubiquitin signals autophagic degradation of cytosolic proteins and peroxisomes

Autophagy is responsible for nonspecific, bulk degradation of cytoplasmic components. Recent work has revealed also that there is specific, autophagic degradation of polyubiquitinated protein aggregates, whose buildup occurs during neurodegenerative disease. Here, we report that simple mono-ubiquitination of normally long-lived cytoplasmic substrates is sufficient to target these substrates for autophagic degradation in mammalian cells. That is, upon their ubiquitination, both small [i.e., red fluorescent protein (RFP)] and large (i.e., peroxisomes) substrates are efficiently targeted to autophagosomes and then degraded within lysosomes upon autophagosome-lysosome fusion. This targeting requires the ubiquitin-binding protein, p62, and is blocked by the Class III phosphatidylinositol 3-kinase (PI3K) inhibitor, 3-methyladenine (3-MA), or by depletion of the autophagy-related-12 (Atg12) protein homolog. Mammalian cells thus use a common pathway involving ubiquitin and p62 for targeting diverse types of substrates for autophagy.

Fluorescence resonance energy transfer using color variants of green fluorescent protein.

Publisher Summary This chapter discusses the fluorescence resonance energy transfer (FRET) using color variants of green fluorescent protein (GFP). Live cell FRET detection in yeast is in its infancy, and there are significant complications with the cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) pair. Spectral overlap between excitation and emission spectra complicates the analysis. The extreme sensitivity of the current YFP to bleaching constrains image acquisition. The detection of a FRET signal is limited by background cellular autofluorescence and the relatively weak fluorescent signal intensities. Dynamic intracellular conditions—such as changes in pH or protein concentrations—can complicate the interpretation of experimental data. However, with careful controls, FRET is a powerful indication of protein–protein interaction. In instances where interactions give robust FRET signals, FRET is a valuable tool used to study the dynamic spatial and temporal behavior of protein–protein interactions in living cells. Future improvements in the spectral properties of CFP and YFP will increase the general applicability of FRET to study a broad range of protein–protein interactions in yeast.

ER–Mitochondrial Calcium Flow Underlies Vulnerability of Mechanosensory Hair Cells to Damage

Mechanosensory hair cells are vulnerable to environmental insult, resulting in hearing and balance disorders. We demonstrate that directional compartmental flow of intracellular Ca2+ underlies death in zebrafish lateral line hair cells after exposure to aminoglycoside antibiotics, a well characterized hair cell toxin. Ca2+ is mobilized from the ER and transferred to mitochondria via IP3 channels with little cytoplasmic leakage. Pharmacological agents that shunt ER-derived Ca2+ directly to cytoplasm mitigate toxicity, indicating that high cytoplasmic Ca2+ levels alone are not cytotoxic. Inhibition of the mitochondrial transition pore sensitizes hair cells to the toxic effects of aminoglycosides, contrasting with current models of excitotoxicity. Hair cells display efficient ER–mitochondrial Ca2+ flow, suggesting that tight coupling of these organelles drives mitochondrial activity under physiological conditions at the cost of increased susceptibility to toxins.

Disruption of Intracellular Calcium Regulation Is Integral to Aminoglycoside-Induced Hair Cell Death

Intracellular Ca2+ is a key regulator of life or death decisions in cultured neurons and sensory cells. The role of Ca2+ in these processes is less clear in vivo, as the location of these cells often impedes visualization of intracellular Ca2+ dynamics. We generated transgenic zebrafish lines that express the genetically encoded Ca2+ indicator GCaMP in mechanosensory hair cells of the lateral line. These lines allow us to monitor intracellular Ca2+ dynamics in real time during aminoglycoside-induced hair cell death. After exposure of live larvae to aminoglycosides, dying hair cells undergo a transient increase in intracellular Ca2+ that occurs shortly after mitochondrial membrane potential collapse. Inhibition of intracellular Ca2+ elevation through either caged chelators or pharmacological inhibitors of Ca2+ effectors mitigates toxic effects of aminoglycoside exposure. Conversely, artificial elevation of intracellular Ca2+ by caged Ca2+ release agents sensitizes hair cells to the toxic effects of aminoglycosides. These data suggest that alterations in intracellular Ca2+ homeostasis play an essential role in aminoglycoside-induced hair cell death, and indicate several potential therapeutic targets to stem ototoxicity.

Functional Mechanotransduction Is Required for Cisplatin-Induced Hair Cell Death in the Zebrafish Lateral Line

Cisplatin, one of the most commonly used anticancer drugs, is known to cause inner ear hair cell damage and hearing loss. Despite much investigation into mechanisms of cisplatin-induced hair cell death, little is known about the mechanism whereby cisplatin is selectively toxic to hair cells. Using hair cells of the zebrafish lateral line, we found that chemical inhibition of mechanotransduction with quinine and EGTA protected against cisplatin-induced hair cell death. Furthermore, we found that the zebrafish mutants mariner (myo7aa) and sputnik (cad23) that lack functional mechanotransduction were resistant to cisplatin-induced hair cell death. Using a fluorescent analog of cisplatin, we found that chemical or genetic inhibition of mechanotransduction prevented its uptake. These findings demonstrate that cisplatin-induced hair cell death is dependent on functional mechanotransduction in the zebrafish lateral line.

There and Back Again: Development and Regeneration of the Zebrafish Lateral Line System

The zebrafish lateral line is a sensory system used to detect changes in water flow. It is comprised of clusters of mechanosensory hair cells called neuromasts. The lateral line is initially established by a migratory group of cells, called a primordium, that deposits neuromasts at stereotyped locations along the surface of the fish. Wnt, FGF, and Notch signaling are all important regulators of various aspects of lateral line development, from primordium migration to hair cell specification. As zebrafish age, the organization of the lateral line becomes more complex in order to accommodate the fishs increased size. This expansion is regulated by many of the same factors involved in the initial development. Furthermore, unlike mammalian hair cells, lateral line hair cells have the capacity to regenerate after damage. New hair cells arise from the proliferation and differentiation of surrounding support cells, and the molecular and cellular pathways regulating this are beginning to be elucidated. All in all, the zebrafish lateral line has proven to be an excellent model in which to study a diverse array of processes, including collective cell migration, cell polarity, cell fate, and regeneration. WIREs Dev Biol 2015, 4:1–16. doi: 10.1002/wdev.160

Fluorescent aminoglycosides reveal intracellular trafficking routes in mechanosensory hair cells

Aminoglycosides (AGs) are broad-spectrum antibiotics that are associated with kidney damage, balance disorders, and permanent hearing loss. This damage occurs primarily by killing of proximal tubule kidney cells and mechanosensory hair cells, though the mechanisms underlying cell death are not clear. Imaging molecules of interest in living cells can elucidate how molecules enter cells, traverse intracellular compartments, and interact with sites of activity. Here, we have imaged fluorescently labeled AGs in live zebrafish mechanosensory hair cells. We determined that AGs enter hair cells via both nonendocytic and endocytic pathways. Both routes deliver AGs from the extracellular space to lysosomes, and structural differences between AGs alter the efficiency of this delivery. AGs with slower delivery to lysosomes were immediately toxic to hair cells, and impeding lysosome delivery increased AG-induced death. Therefore, pro-death cascades induced at early time points of AG exposure do not appear to derive from the lysosome. Our findings help clarify how AGs induce hair cell death and reveal properties that predict toxicity. Establishing signatures for AG toxicity may enable more efficient evaluation of AG treatment paradigms and structural modifications to reduce hair cell damage. Further, this work demonstrates how following fluorescently labeled drugs at high resolution in living cells can reveal important details about how drugs of interest behave.

Robust regeneration of adult zebrafish lateral line hair cells reflects continued precursor pool maintenance

We have examined lateral line hair cell and support cell maintenance in adult zebrafish when growth is largely complete. We demonstrate that adult zebrafish not only replenish hair cells after a single instance of hair cell damage, but also maintain hair cells and support cells after multiple rounds of damage and regeneration. We find that hair cells undergo continuous turnover in adult zebrafish in the absence of damage. We identify mitotically-distinct support cell populations and show that hair cells regenerate from underlying support cells in a region-specific manner. Our results demonstrate that there are two distinct support cell populations in the lateral line, which may help explain why zebrafish hair cell regeneration is extremely robust, retained throughout life, and potentially unlimited in regenerative capacity.

Using the zebrafish lateral line to uncover novel mechanisms of action and prevention in drug-induced hair cell death

The majority of hearing loss and balance disorders are caused by the permanent loss of mechanosensory hair cells of the inner ear. Identification of genes and compounds that modulate susceptibility to hair cell death is frequently confounded by the difficulties of assaying for such complex phenomena in mammalian models. The zebrafish has emerged as a powerful animal model for genetic and chemical screening in many contexts. Several characteristics of the zebrafish, such as its small size and external location of mechanosensory hair cells within the lateral line sensory organ, uniquely position it as an ideal model organism for the study of hair cell toxicity. We have used this model to screen for genes and compounds that affect hair cell survival during ototoxin exposure and have identified agents that would not be expected to play a role in this process based on a priori knowledge of their function. The identification of such agents yields better understanding of hair cell death and holds promise to stem hearing loss and balance disorders in the human population.