The TBC/RabGAP Armus Coordinates Rac1 and Rab7 Functions during Autophagy

Autophagy is an evolutionarily conserved process that enables catabolic and degradative pathways. These pathways commonly depend on vesicular transport controlled by Rabs, small GTPases inactivated by TBC/RabGAPs. The Rac1 effector TBC/RabGAP Armus (TBC1D2A) is known to inhibit Rab7, a key regulator of lysosomal function. However, the precise coordination of signaling and intracellular trafficking that regulates autophagy is poorly understood. We find that overexpression of Armus induces the accumulation of enlarged autophagosomes, while Armus depletion significantly delays autophagic flux. Upon starvation-induced autophagy, Rab7 is transiently activated. This spatiotemporal regulation of Rab7 guanosine triphosphate/guanosine diphosphate cycling occurs by Armus recruitment to autophagosomes via interaction with LC3, a core autophagy regulator. Interestingly, autophagy potently inactivates Rac1. Active Rac1 competes with LC3 for interaction with Armus and thus prevents its appropriate recruitment to autophagosomes. The precise coordination between Rac1 and Rab7 activities during starvation suggests that Armus integrates autophagy with signaling and endocytic trafficking

Ajuba is required for Rac activation and maintenance of E-cadherin adhesion

A Rac–PAK1–Ajuba feedback loop stabilizes cadherin complexes via coordination of spatiotemporal signaling with actin remodeling at cell–cell contacts

Measurement of Electroretinograms and Visually Evoked Potentials in Awake Moving Mice

<div><p>The development of new treatments for intractable retinal diseases requires reliable functional assessment tools for animal models. <i>In vivo</i> measurements of neural activity within visual pathways, including electroretinogram (ERG) and visually evoked potential (VEP) recordings, are commonly used for such purposes. In mice, the ERG and VEPs are usually recorded under general anesthesia, a state that may alter sensory transduction and neurotransmission, but seldom in awake freely moving mice. Therefore, it remains unknown whether the electrophysiological assessment of anesthetized mice accurately reflects the physiological function of the visual pathway. Herein, we describe a novel method to record the ERG and VEPs simultaneously in freely moving mice by immobilizing the head using a custom-built restraining device and placing a rotatable cylinder underneath to allow free running or walking during recording. Injection of the commonly used anesthetic mixture xylazine plus ketamine increased and delayed ERG oscillatory potentials by an average of 67.5% and 36.3%, respectively, compared to unanesthetized mice, while having minimal effects on the a-wave and b-wave. Similarly, components of the VEP were enhanced and delayed by up to 300.2% and 39.3%, respectively, in anesthetized mice. Our method for electrophysiological recording in conscious mice is a sensitive and robust means to assess visual function. It uses a conventional electrophysiological recording system and a simple platform that can be built in any laboratory at low cost. Measurements using this method provide objective indices of mouse visual function with high precision and stability, unaffected by anesthetics.</p></div

Comparison of OPs extracted from ERGs acquired under awake and anesthetized conditions.

<p>Representative ERG traces before and after the extraction of OPs from a scotopic ERG (0 log cd s/m²). Schematic illustration showing the amplitude (a_n) and implicit time (t_n) of each OP wavelet (OP_n). C, D. Amplitudes (C) and implicit times (D) of individual OP wavelets (OP₁–OP₄) recorded in the awake (black) and anesthetized (red) condition from the same mice. E, F. Summed amplitudes (C) and implicit times (D) of all OP wavelets (OP₁–OP₄) from ERGs recorded under awake (black) and anesthetized (red) conditions from the same mice. * <i>P</i> < 0.05. Data from seven mice. The bars indicate mean ± S.E.M. anes: anesthetized.</p

Stability of the ERG and VEP recording.

<p>A, B, Amplitude time courses of ERG and VEP waveforms recorded in anesthetized mice (<i>N</i> = 4 for each time point). The flashes (0 log cd/m²) were given in the scotopic condition. The bars indicate mean ± S.E.M.</p

Comparison of VEPs recorded under awake and anesthetized conditions in the same mice.

<p>Representative traces of scotopic (top) and photopic (bottom) VEPs from the same mouse recorded in the awake (left) and anesthetized (right) condition. Amplitudes of scotopic (top) and photopic (bottom) VEPs from the same mice recorded in the awake (black) and anesthetized (red) condition. Implicit times of scotopic (top) and photopic (bottom) ERGs from the same mice recorded in the awake (black) and anesthetized (red) condition. * <i>P</i> < 0.05. Data from seven mice. The bars indicate mean ± S.E.M. anes: anesthetized.</p