Biology subjects

Barnaba, C.

Publications and source records attributed to Barnaba, C..

3 recordsLinked to original sources

bioRxiv2023

AMPK Regulates Phagophore-to-Autophagosome Maturation

Autophagy is an important metabolic pathway that can non-selectively recycle cellular material or lead to targeted degradation of protein aggregates or damaged organelles. Autophagosome formation starts with autophagy factors accumulating on lipid vesicles containing ATG9. These phagophores attach to donor membranes, expand via ATG2-mediated lipid transfer, capture cargo, and mature into autophagosomes, ultimately fusing with lysosomes for their degradation. Autophagy can be activated by nutrient stress, for example by a reduction in the cellular levels of amino acids. In contrast, how autophagy is regulated by low cellular ATP levels via the AMP-activated protein kinase (AMPK), an important therapeutic target, is less clear. Using live-cell imaging and an automated image analysis pipeline, we systematically dissect how nutrient starvation regulates autophagosome biogenesis. We demonstrate that glucose starvation downregulates autophagosome maturation by AMPK mediated inhibition of phagophores tethering to donor membranes. Our results clarify AMPKs regulatory role in autophagy and highlight its potential as a therapeutic target to reduce autophagy.

cell biology↗

bioRxiv2022

Systematic Analysis of Autophagy Identifies Atg9 Vesicles as the Origin of the Phagophore

Autophagy is a catabolic pathway required for the clearance and recycling of cytoplasmic materials. Upregulation and dysfunction of autophagy contributes to the pathology of cancer and neurogenerative diseases, respectively. To define the molecular mechanisms that control autophagic flux it is critical to quantitatively characterize the dynamic behavior of autophagy factors in living cells. Using a panel of 9 cell lines expressing HaloTagged autophagy factors from their endogenous loci, we systematically analyze the abundance, single-molecule dynamics, and autophagosome association kinetics of a wide variety of autophagy proteins involved in the initiation and maturation of the autophagosome. Our results reveal that phagophores are initiated by the accumulation of autophagy factors on mobile ATG9 vesicles and tethering of these ATG9 vesicles to donor membranes by ATG2 is a key step in phagophore maturation. In addition, we demonstrate that the overall lifetime of an autophagosome is approximately 160 seconds and the majority of phagophore initiation events fail to produce mature autophagosomes. In total our work establishes a new experimental framework to quantitatively analyze autophagy and demonstrates that ATG9 vesicles are the seeds for autophagosome formation in human cells.

cell biology↗

bioRxiv2020

Functional Characterization of an Electromagnetic Perceptive Protein

Magnetoreception, the response to geomagnetic fields is a well described phenomenon in nature. However, it is likely that convergent evolution led to different mechanisms in different organisms. One intriguing example is the unique Electromagnetic Perceptive Gene (EPG) from the glass catfish Kryptopterus vitreolus, that can remotely control cellular function, upon magnetic stimulation in in-vitro and in-vivo. Here, we report for the first time the cellular location and orientation of the EPG protein. We utilized a differential labelling technique to determine that the EPG protein is a membrane anchored protein with an N-terminal extracellular domain. The kinetics and diffusion dynamics of the EPG protein in response to magnetic stimulation was also elucidated using single particle imaging and tracking. Pulse chase labelling and Total Internal Reflection Fluorescence (TIRF) imaging revealed an increase in EPG kinetics post magnetic activation at a single particle level. Trajectory analysis show notably different EPG protein kinetics before and after magnetic stimulation in both 2 (free vs bound particle) and 3 state (free vs intermediate vs bound particle) tracking models. This data provides additional information to support and understand the underlying biophysical mechanisms behind EPG activation by magnetic fields and provides evidence for the basis of magnetoreception in the EPG protein that will aid in future studies that seek to further understand this novel mechanism. This study is important for understanding magnetoreception as well as developing new technologies for magnetogenetics - the utilization of electromagnetic fields to remotely control cellular function. Table of Contents Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/329946v2_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@1ce4aborg.highwire.dtl.DTLVardef@e8bb89org.highwire.dtl.DTLVardef@1705113org.highwire.dtl.DTLVardef@dadf23_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗