Cells transport substances by encasing them in membrane bubbles called vesicles that navigate to various locations within the cell. These vesicles merge with other vesicles to release their contents, a complex process requiring the seamless connection of two membranes without rupturing or leaking. Scientists have long theorized that during this fusion, the cell membrane enters a transient intermediate state, but direct visualization of this process within intact cells has remained elusive until now.

Researchers from the NIH and the University of Virginia embarked on a study to determine if the membranes of living cells create stable, observable structures that signify this intermediate state. They cultured multiple mammalian cell types, including those from humans, monkeys, mice, and rats, in nutrient-rich solutions within laboratory flasks kept in a 37°C (98.6°F) incubator to sustain their growth.

The research team placed between 80,000 and 100,000 cells on a specialized gold-coated platform optimized for high-resolution imaging. To maintain the natural state of the cells, they flash-froze them to immobilize the membranes. Subsequently, they employed a technique known as cryogenic electron tomography to generate detailed images referred to as tomographic images.

Using these cross-sectional images, they reconstructed a 3D model of the cells at the nanometer scale, allowing visibility into the delicate structures of internal vesicles and the plasma membrane. Approximately 300 3D reconstructions showcased areas where membrane bubbles interacted and moved, particularly focusing on membrane contact sites where two vesicles or one vesicle and the cell’s plasma membrane are closely aligned.

Typically, a cell membrane comprises two layers of fat-like molecules that create a flexible barrier. However, the researchers uncovered an uncharacterized membrane structure formed when the outer layers of two membranes merge into a continuous sheet while keeping the inner layers separate. They identified a flat, circular area where the outer layers contacted, forming a thin membrane bridge between vesicles, analogous to soap bubbles merging. This structure is referred to as a hemifome.

The research team noted that hemifsomes are considerably larger and more stable than the ephemeral intermediate states posited by earlier studies. They interpreted this stability to suggest that hemifsomes represent more than mere temporary fusion events; they may endure long enough to engage in vital cellular functions.

Additionally, they detected that some hemifsomes contained singular lens-shaped droplets within the membrane at the fusion point of the two vesicles. About half of the 308 cross-sectional images they analyzed revealed these droplets, averaging 40 nanometers in diameter—approximately 100 times smaller than the adjacent vesicles—and positioned close to the oily membrane interior.

These droplets, distinct from surrounding membrane lipids, are believed to consist of a blend of lipids and proteins, referred to as proteolipid nanodroplets. The researchers posited that the consistent association between hemifsomes and these proteolipid nanodroplets might contribute to the stabilization of hemifsomes or influence the morphological organization of the cell membrane.

To investigate whether hemifsomes facilitate material movement within cells, the team introduced 5- or 15-nanometer-sized gold particles into the cells. These particles were adequately small to traverse the cell’s internal transport systems, which usually distribute nutrients and other molecules. By employing a powerful microscope, they tracked the movement of the gold particles through the cell’s compartments; however, none entered hemifsomes, suggesting a non-involvement in cellular transport.

In conclusion, the researchers posited that hemifsomes emerge when cell membranes merge or reshape, akin to temporary construction sites for cellular membrane construction, repair, or rearrangement. Unlike existing models of membrane fusion and vesicle formation, these findings indicate that vital intermediate states can develop into stable and functional cellular configurations.

The researchers propose that future studies should delve into the molecular composition of proteolipid nanodroplets and clarify how cells regulate the shift from hemifsomes to fully fused membranes. They also recommend exploring hemifsomes’ roles in vesicle formation, membrane recycling, or stress responses across various cell types.

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