Actin dynamics switches two distinct modes of endosomal fusion in yolk sac visceral endoderm cells

Reviewer #1 (Public Review):

Summary:

This manuscript employs yolk sac visceral endoderm cells as a novel model for studying endosomal fusion, observing two distinct fusion behaviors: quick homotypic fusion between late endosomes, and slower heterotypic fusion between late endosomes and lysosomes. The mathematical modeling suggests that vesicle size critically influences the mode of fusion. Further investigations reveal that actin filaments are dynamically associated with late endosomal membranes, and are oriented in the x-y plane and along the apical-basal axis. Actin and Arf2/3 were shown to appear at the rear end of the endosomes along the moving direction suggesting polymerization of actin may provide force for the movement of endosomes. Additionally, the authors found that actin dynamics regulate homotypic and heterotypic fusion events in a different manner. The authors also provide evidence to suggest that Cofilin-dependent actin dynamics are involved in late endosome fusion.

Strengths:

The unique feature of this study is that the authors use yolk sac visceral endoderm cells to study endosomal fusion. Yolk sac visceral endoderm cells have huge endocytic vesicles, endosomes, and lysosomes, offering an excellent system to explore endosomal fusion dynamics and the assembly of cellular factors on membranes. The manuscript provides a valuable and convincing observation of the modes of endosomal fusion and the roles of actin dynamics in this process, and the conclusions of the study are justified by the data.

Weaknesses:

While the study offers compelling observations, it falls short of delivering clear mechanistic insights. Key questions remain unaddressed, such as the functional significance of actin filaments that extend apically in positioning late endosomes, the ways in which actin dynamics influence fusion events, and the functional implications of the slower bridge fusion process.

Reviewer #2 (Public Review):

Summary:

Seiichi Koike et al. studied two fusion models, explosive fusion, and bridge fusion, utilizing yolk sac visceral endoderm cells. They elucidated these two fusion models in vivo by employing mathematical modeling and incorporating fluctuations derived from actin dynamics as a key regulator for rapid homotypic fusion between late endosomes.

Strengths:

This study uncovered the role of actin dynamics in regulating the transition of fusion models in homotypic fusion between late endosomes and introduced a method for observing the fusion of single vesicles with two different targets. The role of actin dynamics in vesicle fusion in other systems has been extensively studied. This study could offer useful insights for research on vesicle fusion.

Weaknesses:
The physiological significance of different fusion models is lacking.

Reviewer #3 (Public Review):

Summary:

The authors found two endosomal fusion modes by live cell imaging of endosomes in yolk sac lateral endoderm cells of 8.5-day-old embryonic mice and described the fusion modes by mathematical models and simulations. They also showed that actin polymerization is involved in the regulation of one of the fusion modes.

Strengths:

The strength of this study is that the authors' claims are well supported by beautiful live cell images and theoretical models. By using specialized cells, yolk sac visceral endoderm cells, the live images of endosomal fusion, localization of actin-related molecules, and validation data from multiple inhibitor experiments are clear.

Weaknesses:

This study does not include any assessment of whether the two types of endosome fusions claimed by the authors occur in general cells, so the article is limited to showing a phenomenon specific to yolk sac lateral endoderm cells. Also, the study does not show the physiological importance of the two types of fusion. There are some unclear points in the method of image analysis and some of the descriptions in the text are not logical.