Fig 1: Western blot analysis of LC3B, septin-3 and p62 protein level changes in primary neurons presented as mean ± SEM due to treatments, with corresponding bands. A Effect of 100 µM AUTEN-67 treatment compared to untreated controls: significant changes in levels of LC3B-II (p = 0.0006), septin-3 (p = 0.014) and p62 (p = 0.010) can be observed. B Effect of 100 µM AUTEN-67, 100 nM Bafilomycin A1 treatment compared to untreated controls: significant changes in levels of LC3B-II (p = 0.034), septin-3 (p = 0.043) and p62 (p = 0.026) can be observed. C Effect of 100 µM AUTEN-67, 10 nM 3-MA treatment compared to controls: no significant change in protein levels can be observed (p = 0.586 for LC3B-II, p = 0.231 for septin-3 and p = 0.0468 for p62)
Fig 2: Protein–protein interaction measurements of septin-3 (SEPT3) and Atg8 homologs. A Microscale thermophoresis dose–response curves of LC3B-SEPT3 (yellow) and GABARAPL2-SEPT3 interactions (blue). X axis represents SEPT3 concentrations on a log scale, while Y axis reflects MST dose–response, normalized to baseline (Fnorm). Corresponding KD values and KD confidence are displayed. B Spectral shift fluorescence response of LC3B-SEPT3 (yellow) and GABARAPL2-SEPT3 (blue). C Fluorescence polarization direct titration curves of LC3B-SEPT3 (marked yellow) and GABARAPL2-SEPT3 (marked blue) interactions. X axis represents SEPT3 concentrations on a linear scale, while Y axis reflects polarization
Fig 3: Trp fluorescence of septin-3. A Structure and sequence of septin-3, marking positions of tryptophans. Core LIR4 and corresponding W284 is marked light-blue, while LIR1 W139 is marked dark-blue. Non-LIR W93 is marked orange. B Trp fluorescence spectra of septin-3 (orange), septin-3-LC3B complex (turquoise), septin-3 after adding 200 nM NaI (grey), septin-3 in complex with LC3B after adding 200 nM NaI (pink)
Fig 4: Confocal microscopy images of primary neuronal cells. A Representative image of enriched LC3B positive particles in the cell body of an AUTEN-67 treated primary neuron indicate enhanced autophagy compared to control. B Overlapping spots of fluorescent signals along the axons (left) and in the cell body (right). C Enlarged and resolved images of overlapping signals from B, with observable LC3B and septin-3 (SEPT3) colocalization, where SEPT3’s colocalization with PINK1 is minimal. However, more complex colocalization patterns of LC3B–PINK1–SEPT3 can be observed from other parts of the sample (D)
Fig 5: Theories of septin-3 function. A Hypothetical roles of septin-3 in synaptic/neuronal autophagy. (1) Septin-3 acting as an adapter between autophagophore-bound LC3B and selective presynaptic cargo escorting it for autophagic degradation. (2) Septin-3 binding to partner motor protein myosin 1B could play role in autophagosome-positioning, or autolysosome biogenesis, since MYO1B is located on lysosomes. (3) TR?B is located on late endosomes and signaling endosomes. Septin-3 is reported binding TR?B. Septin-3 therefore could play a role in autophagosome?late endosome interaction. (4) CDC10, orthologue of septin-3 in yeast localizes to endosomes and autophagosomes and is assumed to play role in membrane-transport to autophagosome. This assumption stands for septin-3 in mammalian neurons. B Possible connections of septin-3 accumulation, neuro-immunological pruning and autophagy
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