Biology subjects

Bakke, O.

Publications and source records attributed to Bakke, O..

4 recordsLinked to original sources

bioRxiv2026

SACHET: seeded contour tracking measures Rab conversion kinetics on individual endosomes in live cells

Membrane-labeled endosomes appear as hollow rings in a projection; they move, deform and exchange their coat, which population trackers (tools that detect every object of one kind and link the detections across frames) are not designed to follow. Here we introduce SACHET (Seeded Adaptive Contour & Hollow Endosome Tracking), a human-supervised Fiji tool that tracks one endosome at a time. On simulated ground truth it localizes the center to 10 nm, recovers transition time constants and lags without bias and holds the target among touching neighbors; comparisons with tuned TrackMate and Cellpose delineate where each is superior. In crowded spinning-disk projections, TrackMate followed one of seven marked Rab5-to-Rab7 conversions; supervised SACHET followed all 37. SACHET thus provides a stable per-endosome frame in which membrane morphology and protein exchange can be quantified: in five cell lines the conversion toggles like a digital switch within seconds to minutes, differing between lines.

cell biology↗

bioRxiv2020

De novo formation of an early endosome during Rab5 to Rab7 transition

Rab5 and Rab7a are the main determinants of early and late endosomes and are important regulators of endosomal progression. The transport from early endosomes to late endosome seems to be regulated through an endosomal maturation switch where Rab5 is exchanged with Rab7a on the same endosome. Here we provide new insight into the mechanism of endosomal maturation where we have discovered a stepwise Rab5 detachment, sequentially regulated by Rab7a. The initial detachment of Rab5 is Rab7a independent and demonstrate a diffusion-like exchange between cytosol and endosomal membrane, and the second phase is slower where Rab5 converges into a specific domain that specifically detaches as a Rab5 indigenous endosome. Consequently, we show that early endosomal maturation regulated through the Rab5 to Rab7a switch induce the formation of a new fully functional early endosome. Progression through a stepwise early endosomal maturation regulates the direction of the transport and concomitantly regulates the homeostasis of early endosomes.View Full Text

cell biology↗

bioRxiv2020

Invariant chain with an AP3 interacting sorting signal is sorted to late endosomal compartments and may improve MHC class I loading and presentation.

Invariant chain (Ii) is traditionally known as the dedicated MHCII chaperone. Recent reports have broadened our understanding about various tasks that Ii plays including its physiological role in MHCI cross-presentation. Ii bound MHCI via the MHCII scaffolding CLIP peptide may facilitate MHCI trafficking to the endosomal pathway. The sorting function of Ii depends on two leucine-based sorting signals present in the cytoplasmic tail that acts as binding sites for the adaptor proteins AP-1/AP-2. Here we increased the Ii cross-presentation potency by replacing these with an AP3 motif resulting an efficient transport of Ii from TGN to late endosomes. We also replaced the CLIP region of li with a therapeutically relevant peptide, MART-1. We found the Ii AP3mutant-MART1 construct was capable of loading MHCI and stimulate specific T-cell response more efficiently than the wild type counterpart. The results show that Ii with an AP3 binding sorting motif carrying peptide epitope(s) can promote efficient antigen presentation to cytotoxic T cells (CTLs) independent of the ER located classical MHCI peptide loading machinery.

immunology↗

bioRxiv2019

Invariant chain regulates endosomal fusion and maturation through the SNARE Vti1b

Invariant chain (Ii) is an important multifunctional player in the regulation of adaptive immune responses and is responsible for several cellular functions related to MHCI and MHCII antigen loading and antigen presentation. While regulating endosomal trafficking of MHCII and other proteins that bind to Ii, this molecule is able to influence the endosomal pathway delaying the maturation of endosomes to the late endosomal loading compartments. When expressed in cells Ii is found to increase endosomal size, but the mechanisms for this is not known. We used RNAi silencing to identify SNARE proteins controlling Ii induced increase of endosomal size and delay of the endosomal pathway. Ii was found to interact with the SNARE protein Vti1b. Vti1b localized at the contact sites of fusing Ii positive endosomes and a tailless Ii was able to relocate Vti1b to the plasma membrane. Furthermore, silencing Vti1b, abrogated the delay in endosomal maturation induced by Ii expression. In conclusion, Ii interacts with Vti1b and this interaction is fundamental for Ii-mediated alteration of the endosomal pathway. We propose that Ii, by interacting with SNAREs, in particular Vti1B in the biosynthetic pathway of antigen presenting cells, is able to assemble SNARE directed fusion partners in the early part of the endosomal pathway that lead to a slower endosomal maturation for efficient antigen processing and antigen loading.

cell biology↗