Hemifused Vesicles and Their Role in Cellular Transport

Introduction

Cells constantly remodel the structure of their internal membranes to help traffic and transport proteins, lipids, and other forms of cargo. This happens through essential processes like membrane fusion, scission and intraluminal vesicle formation which are integral to cellular function and maintaining homeostasis. While significant research has been done on the mechanisms through which these processes occur, the direct, in-situ visualization and characterization of the process and its intermediate structures has been a remarkable challenge.

Additionally, the precise mechanisms through which membrane remodelling happens to ensure proper trafficking and transport of membrane proteins and lipids, which maintains cellular health and function remains a topic of broad interest in researchers.

Recent advances in a particular visualization method called in-situ cryo-electron tomography (cryo-ET) has enabled the gain of invaluable insights on cellular structures in near-native states. One of the largest discoveries made through this is the discovery of a new class of organelles within the cell, called hemifusomes, which are characterized by two heterotypic vesicles hemifused (partially fused) together.

Method

1. The mammalian cells were cultured in a medium created from a mix of nutrient serums, and were placed in a gold EM grid. 

2. The cells were then flash frozen using liquid ethane at -176°C

3. Flash freezing prevents the formation of any ice crystals

3. No chemical staining was used to ensure the cells stay at its most natural state, as chemical interference can cause distortion

4. The frozen cell sample was placed under an electron microscope

5. The sample was tilted and imaged many times

6. The 2D images were computationally combined to form a 3D image (tomogram)

The periphery was focused on the edges of cells, where the trafficking of vesicles is very high

Results

Through the tomographs, scientists were able to capture images of the thin cellular regions, including ribosome associated vesicles (RAVs), endosomes, lysosomes, and multivesicular bodies (MVBs), which were of particular interest.

The images provided above are few out of the many taken during the experiment. They revealed numerous heterotypic pairs of vesicles connected by a shared membrane region - the Hemifusion Diaphragm (HD), a unique conformation previously thought to be too unstable to serve any purpose other than act as an intermediate for vesicular fusion or scission

Through this study, it was found that Hemifusomes (HF) are found in two conformations:

1. Direct Hemifusomes - a small vesicle is paired to the cytoplasmic side of a larger vesicle

2. Flipped Vesicles (fHF) - a vesicle is hemifused to the inner side of the membrane bilayer of a larger vesicle

Additionally, 3D tomographs frequently revealed a dense particle, or nanodroplet, integrated into the bilayer at the margin of the HD

Comparison of the hemifusome luminal content to the content of other membrane-bound organelles

The lumen of the larger vesicle within the heterotrophic vesicles was found to have a fine granular texture, similar to RAVs and endosomes. Meanwhile, the unique smooth and translucent appearance of the smaller vesicles was found to be unique and did not match the texture or electron density of any other vesicular organelles, including endosome-like vesicles, clathrin-coated vesicles, RAVs, lipid droplets, and various conformations of endosomes. These vesicles were not found free or docked to any vesicles other than hemifusomes within the whole cell. 

These findings led to the question of whether hemifusomes might be formed from alternate mechanisms to normal vesicular fusion.

Morphology and Variability

Representative close-up views of the rims of the hemifusome HDs, where the bilayer of the two vesicles and the HD meet, enabled researchers to confirm that the membrane bilayer of both vesicles were indeed in contact, with the shared HD being comparable to typical membranes at ~4 nm. The paired vesicles forming the hemifusome can vary largely in both their independent and relative sizes. Even when the vesicles were of similar size, they displayed large variation in their radius and curvature, indicating diverse degrees of radial expansion in the areas of hemifusion.

It was also observed that the shape of the complex could be influenced by physical constraints, such as through surrounding actin filaments and microtubules, or proximity to the plasma membrane. These varying conformations showed the compressibility and deformability of the hemifusome and how internal and external forces or constraints contributed to its overall shape.

The average radius of the larger vesicle was found to be ~300 nm  ± 96.2 nm, while the HDs of the same hemifusomes was found to be around 160 nm. This was found to be much larger than the transient HDs of canonical membrane fusion events. Many times the HD was comparable to the remaining membrane length of the smaller vesicle. In this configuration, the entire inner contents of the smaller vesicle are fully encapsulated within the bilayer of the larger vesicle, creating a long-lived, lens-shaped product of hemifusion.

Parallels between Direct and Flipped Hemifusomes

In the 308 tomographs which were taken, scientists were able to clearly identify 88 direct hemifusomes and 48 ones in the flipped configuration. Based on the topological features of both configurations, its highly likely that they both represent two configurations of the same organelle, potentially transitioning from one to the other.

Presence of a Proteolipid Nanodroplet at the Hemifusion Diaphragm

Tomographic analysis of hemifusomes consistently revealed a single dense structure embedded within the hydrophobic interior of the bilayers in a “three-way” junction of the HD and the two heterotrophic vesicles. Detailed views showed this nanodroplet interfacing with the hydrophobic side of the hemifused vesicles, suggesting that it contained hydrophobic components, likely lipids. However the contents of the nanodroplet contained particulate structures, strongly suggesting it contained proteins, leading scientists to conclude that the nanodroplets are of a mixed proteolipid composition.

Limitations in even the best tomograms only allowed pictures to cover only around a third of the hemifusomes circumference, with the top and bottom third cut out of the image. This led to many of the structures being missed during observation. Regardless, a nanodroplet was found in nearly half of the 300 tomographs taken, allowing scientists to strongly presume a frequency of around one nanodroplet per hemifusome, with no instances of two or more nanodroplets being found in the HD. This observation raises the possibility that the insertion of the nanodroplet might trigger the initiation, formation and stabilization of the hemifusome.

Compound Hemifusomes as Hubs for the Formation of Complex Multivesicular Bodies

One quarter of the total hemifusomes found, both direct and flipped, identified one or more additional vesicles attached in a hemifused configuration with either or both of the vesicles of the initial pair. The occurrence of these compound hemifusomes further demonstrates the unexpected longevity of the HDs within the hemifusomes. Scientists also observed multiple instances of additional proteolipid nanodroplets (PNDs) embedded in the membranes. Based on hypothesised models of PNDs as hubs for vesicle biogenesis, scientists propose that additional PNDs are likely sites for the initiation of compound hemifusomes.

The multiple hemifusion events coalesce to form very large complex structures, which can harbor a range of conformations of direct and flipped hemifusomes. Researchers speculate that compound hemifusomes, followed by the flipping of a smaller vesicle into the luminal side of a large one, can provide an alternate path for the formation of intraluminal vesicles.

Discussion 

Through this study, researchers have identified a previously unrecognised vesicular organelle complex with its own membrane topology. This complex, which they have termed “hemifusomes”, consists of hemifused heterotrophic vesicles sharing a large hemifusion diaphragm (HD). The presence of the large, long-lived HDs is particularly surprising, given the widely accepted view that HDs are small, unstable and only occur as transient intermediates in vesicular fusion events. A second intriguing feature was the consistent presence of a ~42 nm proteolipid nanodroplet embedded in the membrane of the hemifused vesicles. This localization suggests a role of PNDs in the formation, stabilization, or expansion of the hemifusion interface.

The paired vesicles were heterotypic in both size and luminal content. Hemifusomes were present in two different configurations: direct, and flipped. Given their distinctive topology and variety of conformations, researchers predict that hemifusomes may play roles in protein and lipid sorting, recycling, and the formation of intraluminal vesicles.

There is a possibility of the hemifusome being created from two independent, pre-existing vesicles. However, the smaller vesicles consistently showed a translucent appearance of the luminal content, which is unique from all other luminal content found in any vesicles. The study contained new observations on the morphology of hemifusomes in-situ, which could happen due to osmotic pressure or high membrane stresses. Analysis also found the HD can stabilize into a lens-shaped complex, using “dead-end hemifusion”. Observation concludes that this could serve a biological function other than serving as an intermediate.

Scientists propose that these lens-shaped structures are long-lived and can evolve into intraluminal vesicles. This process may involve a new method of intraluminal budding distinct from the canonical ESCRT pathway. Given the constant presence of PNDs, scientists hypothesize that they are hubs for hemifusome formation, and that this PND-dependent process, which they term “vesiculogenesis”, represents an alternative pathway from ESCRT-based budding.

The HDs, with their unique bilayer, will impact the conformation and distribution of proteins according to their topological sensitivity. They could be involved in many processes, such as protein sorting, lipid transfer, and lipid sorting.

Open Questions

In-situ cryo-ET is arguably the most promising approach for obtaining native structural information on cellular organelles. The images in this study of clathrin-coated pits and vesicles reveal additional structural features beyond those visualized using normal methods. The study aimed to minimize stress responses in the cells that often occur from sample handling and preparation. This was done by reducing sample manipulation down to the essential steps only. However, while unlikely, it is possible hemifusomes were formed in the brief transfer period between steps in the method. This can be considered a rapid stress response that must be taken into consideration in the evaluation of the in-situ cryo-ET process. It is more likely that hemifusomes were previously overlooked in classical EM due to fixation and dehydration steps, which could alter stability and appearance. 

This study was limited to observation of the thin regions at the cellular periphery. Further research should focus on determining whether they are also found in other regions of the cell, and explaining their molecular mechanisms based on its formation, stability and functions. Additionally, studies should be done on the broader implications of Hemifusomes on cellular physiology and pathology. 

Arya Shiralkar | Writer | The STEM Review