Uncoated Endocytic Vesicles Require the Unconventional Myosin, Myo6, for Rapid Transport through Actin Barriers

After clathrin-mediated endocytosis, clathrin removal yields an uncoated vesicle population primed for fusion with the early endosome. Here we present the first characterization of uncoated vesicles and show that myo6, an unconventional myosin, functions to move these vesicles out of actin-rich regions found in epithelial cells. Time-lapse microscopy revealed that myo6-associated uncoated vesicles were motile and exhibited fusion and stretching events before endosome delivery, processes that were dependent on myo6 motor activity. In the absence of myo6 motor activity, uncoated vesicles remained trapped in the actin mesh, where they exhibited Brownian-like motion. Exit from the actin mesh occurred by a slow diffusion-based mechanism, delaying transferrin trafficking to the early endosome. Expression of a myo6 mutant that bound tightly to F-actin produced immobilized vesicles and blocked trafficking. Depolymerization of the actin cytoskeleton rescued this block and specifically accelerated transferrin delivery to the early endosome without affecting earlier steps in endocytosis. Therefore actin is a physical barrier impeding uncoated vesicle trafficking, and myo6 is recruited to move the vesicles through this barrier for fusion with the early endosome.

Download Full-text

Myo6 Facilitates the Translocation of Endocytic Vesicles from Cell Peripheries

Molecular Biology of the Cell ◽

10.1091/mbc.e02-11-0767 ◽

2003 ◽

Vol 14 (7) ◽

pp. 2728-2743 ◽

Cited By ~ 134

Author(s):

Laura Aschenbrenner ◽

TinThu Lee ◽

Tama Hasson

Keyword(s):

Epithelial Cells ◽

Fluorescent Protein ◽

Early Endosome ◽

Sufficient Time ◽

Significant Delay ◽

Transfected Cells ◽

Early Endosomes ◽

Green Fluorescent ◽

Endocytic Vesicles ◽

Transferrin Uptake

Immunolocalization studies in epithelial cells revealed myo6 was associated with peripherally located vesicles that contained the transferrin receptor. Pulse-chase experiments after transferrin uptake showed that these vesicles were newly uncoated endocytic vesicles and that myo6 was recruited to these vesicles immediately after uncoating. GIPC, a putative myo6 tail binding protein, was also present. Myo6 was not present on early endosomes, suggesting that myo6 has a transient association with endocytic vesicles and is released upon early endosome fusion. Green fluorescent protein (GFP) fused to myo6 as well as the cargo-binding tail (M6tail) alone targeted to the nascent endocytic vesicles. Overexpression of GFP-M6tail had no effect on a variety of organelle markers; however, GFP-M6tail displaced the endogenous myo6 from nascent vesicles and resulted in a significant delay in transferrin uptake. Pulse-chase experiments revealed that transferrin accumulated in uncoated vesicles within the peripheries of transfected cells and that Rab5 was recruited to the surface of these vesicles. Given sufficient time, the transferrin did traffic to the perinuclear sorting endosome. These data suggest that myo6 is an accessory protein required for the efficient transportation of nascent endocytic vesicles from the actin-rich peripheries of epithelial cells, allowing for timely fusion of endocytic vesicles with the early endosome.

Download Full-text

Faculty Opinions recommendation of Uncoated endocytic vesicles require the unconventional myosin, Myo6, for rapid transport through actin barriers.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1005517.211527 ◽

2004 ◽

Author(s):

William Bement

Keyword(s):

Unconventional Myosin ◽

Rapid Transport ◽

Endocytic Vesicles

Download Full-text

Characterization of connective tissue progenitors through phase contrast and multicolor fluorescence time-lapse microscopy

10.1117/12.2077230 ◽

2015 ◽

Author(s):

Edward Kwee ◽

Kimerly Powell ◽

George Muschler

Keyword(s):

Connective Tissue ◽

Phase Contrast ◽

Time Lapse ◽

Time Lapse Microscopy ◽

Multicolor Fluorescence

Download Full-text

Experience of using time-lapse microscopy in IVF and ICSI programs

Medical alphabet ◽

10.33667/2078-5631-2020-16-47-50 ◽

2020 ◽

pp. 47-50

Author(s):

N. V. Saraeva ◽

N. V. Spiridonova ◽

M. T. Tugushev ◽

O. V. Shurygina ◽

A. I. Sinitsyna

Keyword(s):

Pregnancy Rate ◽

Embryo Transfer ◽

Selection Procedure ◽

Time Lapse ◽

Single Embryo Transfer ◽

Main Group ◽

Clinical Pregnancy ◽

Control Group ◽

Time Lapse Microscopy

In order to increase the pregnancy rate in the assisted reproductive technology, the selection of one embryo with the highest implantation potential it is very important. Time-lapse microscopy (TLM) is a tool for selecting quality embryos for transfer. This study aimed to assess the benefits of single-embryo transfer of autologous oocytes performed on day 5 of embryo incubation in a TLM-equipped system in IVF and ICSI programs. Single-embryo transfer following incubation in a TLM-equipped incubator was performed in 282 patients, who formed the main group; the control group consisted of 461 patients undergoing single-embryo transfer following a traditional culture and embryo selection procedure. We assessed the quality of transferred embryos, the rates of clinical pregnancy and delivery. The groups did not differ in the ratio of IVF and ICSI cycles, average age, and infertility factor. The proportion of excellent quality embryos for transfer was 77.0% in the main group and 65.1% in the control group (p = 0.001). In the subgroup with receiving eight and less oocytes we noted the tendency of receiving more quality embryos in the main group (р = 0.052). In the subgroup of nine and more oocytes the quality of the transferred embryos did not differ between two groups. The clinical pregnancy rate was 60.2% in the main group and 52.9% in the control group (p = 0.057). The delivery rate was 45.0% in the main group and 39.9% in the control group (p > 0.050).

Download Full-text