High-Density Lipoproteins (HDL), colloquially known as “good cholesterol,” have long been recognized for their critical role in maintaining cardiovascular health by transporting excess cholesterol from peripheral tissues back to the liver for excretion or recycling. This reverse cholesterol transport mechanism is vital to preventing atherosclerosis—a pathological condition characterized by plaque accumulation within arterial walls. The clinical consequences of atherosclerosis are severe, encompassing heart attacks, strokes, aneurysms, and thrombotic events that collectively represent leading causes of morbidity and mortality worldwide. Despite the well-established physiological importance of HDL, the precise molecular processes that mediate HDL formation have remained enigmatic, impeding progress toward targeted therapies for cholesterol-related diseases.
Conventional thinking held that HDLs remove cellular cholesterol primarily through passive diffusion. However, this paradigm was challenged by genetic insights gleaned from studies of Tangier disease, a rare inherited disorder marked by markedly reduced plasma HDL levels. These studies identified ATP-binding cassette protein A1 (ABCA1), an ATP-dependent transmembrane transporter, as essential for efficient HDL biosynthesis. The discovery raised profound questions about the mechanistic underpinnings of HDL biogenesis: How does ABCA1 harness ATP hydrolysis to mobilize cholesterol and phospholipids? What conformational states does ABCA1 adopt during HDL generation, and how does its extracellular domain participate in this process?
Harnessing cutting-edge high-speed atomic force microscopy (HS-AFM), a research collaboration led by Professor Kazumitsu Ueda at Kyoto University’s Institute for Integrated Cell-Material Sciences (iCeMS) teamed up with experts at Kanazawa University to shed light on these questions. HS-AFM enables real-time visualization of biomolecular activities with nanometer spatial resolution and sub-second temporal precision, an advancement that surpasses classical cryoelectron microscopy by capturing dynamic conformational changes in native-like environments. This unprecedented imaging capability allowed the team to monitor ABCA1’s behavior at the membrane interface during nascent HDL formation, providing an intimate view of lipid transport and complex assembly that had never before been observed.
Initial hypotheses posited that ABCA1’s extracellular domain (ECD) served as a static lipid reservoir, temporarily accommodating approximately 500 cholesterol and phospholipid molecules on its outer face. However, early structural data from cryoelectron microscopy suggested that the ECD forms a narrow tunnel structure, seemingly incongruent with the volume necessary to harbor such a large number of lipids. This discrepancy hinted at a more intricate mechanism involving dynamic structural reorganization. Using HS-AFM, Ueda’s team visualized that rather than sitting passively, the ECD actively undergoes conformational remodeling during lipid translocation, expanding and subsequently reducing its volume by nearly 30% as it loads lipids en masse.
The process begins as ABCA1 hydrolyzes ATP molecules, harnessing the energy released to power the translocation of lipid molecules from the inner leaflet of the plasma membrane through the transmembrane regions and into the ECD. The researchers observed that the ECD temporarily “inflates,” generating novel structural features capable of storing a substantial lipid payload. These lipids are then transferred collectively onto apolipoprotein A-I (apoA-I), a protein that serves as a lipid acceptor and scaffold for HDL particle assembly. This cooperative loading mechanism culminates in the formation of nascent HDL particles, which are subsequently released into circulation to fulfill their cholesterol-scavenging functions.
This newfound insight into the dynamic lipid handling by ABCA1 challenges prior models of HDL formation that relied heavily on simplistic diffusion or tunnel-based lipid transfer. Instead, it illuminates a sophisticated energy-dependent cycle of membrane remodeling and lipid packaging orchestrated by ABCA1’s ATP-driven conformational flexibility. Such a paradigm shift expands our fundamental grasp of cholesterol metabolism and opens new avenues for therapeutic modulation targeting the structural states of ABCA1, potentially enhancing HDL biogenesis or correcting defects seen in dyslipidemic conditions.
Moreover, the HS-AFM methodology employed in this study represents a remarkable technical breakthrough. The ability to perform direct side-view imaging of membrane proteins, particularly large transporters like ABCA1, remains an exceptionally challenging feat. By overcoming these hurdles, the research introduces a versatile platform that can be extended to investigate a broad spectrum of biological transporters involved in lipid trafficking, drug extrusion, and metabolic waste removal, significantly enriching our structural and functional understanding of membrane proteins critical to health and disease.
Professor Ueda emphasizes the clinical and biological ramifications of these findings: “Our detailed visualization of ABCA1-mediated HDL formation helps clarify the physiological roles of HDL and cholesterol, elements often misunderstood in cardiovascular research. By elucidating the regulatory mechanisms that guide ABCA1 function, we anticipate more precise targeting in the development of therapies for cholesterol-related disorders.” This pioneering work thus bridges a long-standing knowledge gap, with the potential to reshape strategies in combating atherosclerosis and related cardiovascular conditions.
In addition to the academic impact, the team demonstrates the power of interdisciplinary collaboration, leveraging the precision of nano-scale imaging with biochemical expertise to tackle complex biological questions that were once inaccessible. The combined efforts of Kyoto University and Kanazawa University scientists accentuate the promise of emerging imaging technologies in unraveling intricate molecular phenomena.
Moving forward, these revelations could inspire innovative therapeutic approaches aimed at modulating ABCA1 activity or stabilizing particular conformational states to optimize HDL generation. Furthermore, understanding the nuanced interplay between “good” HDL cholesterol and “bad” low-density lipoproteins (LDL) within the vascular milieu may eventually yield comprehensive treatments that address the multifactorial nature of cholesterol-driven disease pathogenesis.
This study’s broader contributions extend beyond cardiovascular health; the ability to monitor membrane protein dynamics live and in situ could inform the design of novel interventions across diverse fields including metabolic regulation, drug resistance, and membrane biology. Consequently, the implications of this work resonate well beyond the confines of lipid metabolism, establishing new frontiers in molecular medicine.
The authors have made available supplementary video content capturing the real-time structural dynamics of ABCA1 during lipid transfer, providing compelling visual evidence of these molecular events. These materials not only enhance scientific transparency but also serve as valuable educational tools for the broader research community.
As technology continues to advance, the integration of high-speed atomic force microscopy with complementary biophysical techniques is poised to deepen our mechanistic understanding of cellular transport processes. Ultimately, such insights pave the way for precision medicine strategies tailored to correct or augment fundamental biological pathways at the molecular level.
Subject of Research: Molecular mechanism of HDL biogenesis mediated by ATP-binding cassette protein A1 (ABCA1)
Article Title: Direct Visualization of ATP-Binding Cassette Protein A1 Mediated Nascent High-Density Lipoprotein Biogenesis by High-Speed Atomic Force Microscopy
News Publication Date: 20-Aug-2025
Web References: http://dx.doi.org/10.1021/acs.nanolett.5c03116
References: Kodan et al., Nano Letters, 2025
Image Credits: The Authors
Keywords: Cell biology, Molecular biology, Biochemistry, Atomic force microscopy, Dyslipidemia
