Publications

 

MICHAEL V. KEEBLER

Ph.D.

PUBLICATIONS

The Two-pore channel (TPC) interactome unmasks isoform-specific roles for TPCs in endolysosomal morphology and cell pigmentation.

 

Lin-Moshier Y, Keebler MV, Hooper R, Boulware MJ, Liu X, Churamani D, Abood ME, Walseth TF, Brailoiu E, Patel S, Marchant JS.

Proc Natl Acad Sci U S A. 2014 Sep 9;111(36):13087-92. doi: 10.1073/pnas.1407004111.

 

The two-pore channels (TPC1 and TPC2) belong to an ancient family of intracellular ion channels expressed in the endolysosomal system. Little is known about how regulatory inputs converge to modulate TPC activity, and proposed activation mechanisms are controversial. Here, we compiled a proteomic characterization of the human TPC interactome, which revealed that TPCs complex with many proteins involved in Ca(2+) homeostasis, trafficking, and membrane organization. Among these interactors, TPCs were resolved to scaffold Rab GTPases and regulate endomembrane dynamics in an isoform-specific manner. TPC2, but not TPC1, caused a proliferation of endolysosomal structures, dysregulating intracellular trafficking, and cellular pigmentation. These outcomes required both TPC2 and Rab activity, as well as their interactivity, because TPC2 mutants that were inactive, or rerouted away from their endogenous expression locale, or deficient in Rab binding, failed to replicate these outcomes. Nicotinic acid adenine dinucleotide phosphate (NAADP)-evoked Ca(2+) release was also impaired using either a Rab binding-defective TPC2 mutant or a Rab inhibitor. These data suggest a fundamental role for the ancient TPC complex in trafficking that holds relevance for lysosomal proliferative scenarios observed in disease.

Spatial organization of intracellular Ca2+ signals.

 

Konieczny V, Keebler MV, Taylor CW.

Semin Cell Dev Biol. 2012 Apr;23(2):172-80. doi: 10.1016/j.semcdb.2011.09.006.

 

The ability of Ca2+, the simplest of all intracellular messengers, selectively to regulate so many cellular behaviours is due largely to the complex spatiotemporal organization of intracellular Ca2+ signals. Most signalling pathways, including those that culminate in Ca2+ signals, comprise sequences of protein-protein interactions linked by diffusible messengers. Using specific examples to illustrate key principles, we consider the roles of both components in defining the spatial organization of Ca2+ signals. We discuss evidence that regulation of most Ca2+ channels by Ca2+ contributes to controlling the duration of Ca2+ signals, to signal integration and, via Ca2+-induced Ca2+ release, to defining the spatial spread of Ca2+ signals. We distinguish two types of protein-protein interaction: scaffolds that allow rapid local transfer of diffusible messengers between signalling proteins, and interactions that directly transfer information between signalling proteins. Store-operated Ca2+ entry provides a ubiquitous example of the latter, and it serves also to illustrate how Ca2+ signals can be organized at different levels of spatial organization - from interactions between proteins to interactions between organelles.

Pitch perception beyond the traditional existence region of pitch.

 

Oxenham AJ, Micheyl C, Keebler MV, Loper A, Santurette S.

Proc Natl Acad Sci U S A. 2011 May 3;108(18):7629-34. doi: 10.1073/pnas.1015291108.

 

Humans' ability to recognize musical melodies is generally limited to pure-tone frequencies below 4 or 5 kHz. This limit coincides with the highest notes on modern musical instruments and is widely believed to reflect the upper limit of precise stimulus-driven spike timing in the auditory nerve. We tested the upper limits of pitch and melody perception in humans using pure and harmonic complex tones, such as those produced by the human voice and musical instruments, in melody recognition and pitch-matching tasks. We found that robust pitch perception can be elicited by harmonic complex tones with fundamental frequencies below 2 kHz, even when all of the individual harmonics are above 6 kHz--well above the currently accepted existence region of pitch and above the currently accepted limits of neural phase locking. The results suggest that the perception of musical pitch at high frequencies is not constrained by temporal phase locking in the auditory nerve but may instead stem from higher-level constraints shaped by prior exposure to harmonic sounds.

Musical intervals and relative pitch: frequency resolution, not interval resolution, is special.

 

McDermott JH, Keebler MV, Micheyl C, Oxenham AJ.

J Acoust Soc Am. 2010 Oct;128(4):1943-51. doi: 10.1121/1.3478785.

 

Pitch intervals are central to most musical systems, which utilize pitch at the expense of other acoustic dimensions. It seemed plausible that pitch might uniquely permit precise perception of the interval separating two sounds, as this could help explain its importance in music. To explore this notion, a simple discrimination task was used to measure the precision of interval perception for the auditory dimensions of pitch, brightness, and loudness. Interval thresholds were then expressed in units of just-noticeable differences for each dimension, to enable comparison across dimensions. Contrary to expectation, when expressed in these common units, interval acuity was actually worse for pitch than for loudness or brightness. This likely indicates that the perceptual dimension of pitch is unusual not for interval perception per se, but rather for the basic frequency resolution it supports. The ubiquity of pitch in music may be due in part to this fine-grained basic resolution.

Pitch perception for mixtures of spectrally overlapping harmonic complex tones.

 

Micheyl C, Keebler MV, Oxenham AJ.

J Acoust Soc Am. 2010 Jul;128(1):257-69. doi: 10.1121/1.3372751.

 

This study measured difference limens for fundamental frequency (DLF0s) for a target harmonic complex in the presence of a simultaneous spectrally overlapping harmonic masker. The resolvability of the target harmonics was manipulated by bandpass filtering the stimuli into a low (800-2400 Hz) or high (1600-3200 Hz) spectral region, using different nominal F0s for the targets (100, 200, and 400 Hz), and different masker F0s (0, +9, or -9 semitones) relative to the target. Three different modes of masker presentation, relative to the target, were tested: ipsilateral, contralateral, and dichotic, with a higher masker level in the contralateral ear. Ipsilateral and dichotic maskers generally caused marked elevations in DLF0s compared to both the unmasked and contralateral masker conditions. Analyses based on excitation patterns revealed that ipsilaterally masked F0 difference limens were small (<2%) only when the excitation patterns evoked by the target-plus-masker mixture contained several salient (>1 dB) peaks at or close to target harmonic frequencies, even though these peaks were rarely produced by the target alone. The findings are discussed in terms of place- or place-time mechanisms of pitch perception.

 

 

An ancestral deuterostome family of two-pore channels mediates nicotinic acid adenine dinucleotide phosphate-dependent calcium release from acidic organelles.

 

Brailoiu E, Hooper R, Cai X, Brailoiu GC, Keebler MV, Dun NJ, Marchant JS, Patel S.

J Biol Chem. 2010 Jan 29;285(5):2897-901. doi: 10.1074/jbc.C109.081943.

 

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent and widespread calcium-mobilizing messenger, the properties of which have been most extensively described in sea urchin eggs. The molecular basis for calcium release by NAADP, however, is not clear and subject to controversy. Recent studies have provided evidence that members of the two-pore channel (TPC) family in mammals are the long sought after target channels for NAADP. Here, we show that the TPC3 gene, which has yet to be functionally characterized, is present throughout the deuterostome lineage but is a pseudogene in humans and other primates. We report the molecular cloning of the complete ancestral TPC gene family from the sea urchin and demonstrate that all three isoforms localize to acidic organelles to mediate NAADP-dependent calcium release. Our data highlight the functional divergence of this novel gene family during deuterostome evolution and provide further evidence that NAADP mediates calcium release from acidic stores through activation of TPCs.

Can temporal fine structure represent the fundamental frequency of unresolved harmonics?

 

Oxenham AJ, Micheyl C, Keebler MV.

J Acoust Soc Am. 2009 Apr;125(4):2189-99. doi: 10.1121/1.3089220.

 

This study measured difference limens for fundamental frequency (DLF0s) for a target harmonic complex in the presence of a simultaneous spectrally overlapping harmonic masker. The resolvability of the target harmonics was manipulated by bandpass filtering the stimuli into a low (800-2400 Hz) or high (1600-3200 Hz) spectral region, using different nominal F0s for the targets (100, 200, and 400 Hz), and different masker F0s (0, +9, or -9 semitones) relative to the target. Three different modes of masker presentation, relative to the target, were tested: ipsilateral, contralateral, and dichotic, with a higher masker level in the contralateral ear. Ipsilateral and dichotic maskers generally caused marked elevations in DLF0s compared to both the unmasked and contralateral masker conditions. Analyses based on excitation patterns revealed that ipsilaterally masked F0 difference limens were small (<2%) only when the excitation patterns evoked by the target-plus-masker mixture contained several salient (>1 dB) peaks at or close to target harmonic frequencies, even though these peaks were rarely produced by the target alone. The findings are discussed in terms of place- or place-time mechanisms of pitch perception.

CONTACT DETAILS

 

michael.v.keebler@gmail.com

 

Tel: +44 (0)1223 834244

© 2016 Michael V. Keebler. All rights reserved.