THEME 9 IMAGING IMMUNE EFFECTORS

The cryo-EM structure of the acid activatable pore-forming immune effector Macrophage-expressed gene 1.
Nature Communications, Vol 10, 4288 (2019) Pang SS, Bayly-Jones C, Radjainia M, Spicer BA, Law RHP, Hodel AW, Parsons ES, Ekkel SM, Conroy PJ, Ramm G, Venugopal H, Bird PI, Hoogenboom BW, Voskoboinik I, Gambin Y, Sierecki E, Dunstone MA, Whisstock JC.

“Cryo-EM structures of a Macrophage perforin-like protein in the pre-pore form show how cells can use pore forming proteins to destroy bacteria within an intracellular compartment
without damaging host cell membranes.”

This work uses structural biology and biochemistry to understand how pore forming immune effectors function to destroy targets. In particular, we are interested in three mammalian proteins – Complement component-9 (C9), Macrophage Expressed Gene-1 (MPEG-1) and Perforin. These three molecules play key roles in the destruction of pathogenic microbes, virally infected cells and malignant cells. Concomitant with working on these protein complexes, we are also developing new and better approaches for sample preparation for cryogenic Electron Microscopy experiments and for determining protein structures in the context of intact, cryogenically preserved cells.

PROF. JAMES WHISSTOCK

AT A GLANCE

In 2019, we reported the cryo-EM structure of the novel perforin like protein MPEG-1. This molecule is deployed by macrophages within phagolysosomes in order to destroy engulfed microbial targets. (Pang et al., Nature Communications 2019). The structure represents the first atomic-resolution structure of any perforin-like protein in a bona fide pre-pore intermediate assembly. We also showed that MPEG-1 pre-pores are activated to form pores only at acidic pH, suggesting that acidification of the phagolysosome represents a crucial trigger for MPEG-1 function.

The structure further revealed that MPEG-1 uses an N-terminal multi-vesicular body of 12 kDa (MVB12)-associated β-prism (MABP) domain in order to bind membranes. However, unexpectedly, the MABP domain was positioned such that the membrane-binding face pointed in the opposite orientation to other perforin-like proteins. Collectively, together with liposome binding data (see Hero image), our data suggest that this unusual arrangement of domains permits MPEG-1 to bind to the inner leaflet of the host membrane, with pore formation into pathogenic bacteria taking place in trans. This mechanism would prevent auto-lysis of the macrophage vesicle (See image on page 58), an event that would likely lead to macrophage destruction.

With respect to imaging technologies, our team has been working to develop new approaches to streamline the in situ structural biology workflow. This approach permits visualization of large protein complexes, such as the mega-dalton sized pores formed by perforin-like proteins, in the context of cryo-preserved cells. Most notably this year we reported our development of the Photon Ion Electron microscope (PIE-scope; Gorelick et al., elife, 2019). In this instrument, we have integrated a cryo-Focused Ion Bean Scanning Electron Microscope (cryo FIBSEM) together with a light microscope. This powerful new tool permits fluorescent imaging of sample under cryogenic conditions within the cryo-chamber of a cryo-FIBSEM. This procedure avoids having to move material between several different microscopes, thus greatly reducing ice damage and further permitting rapid, fluorescently targeted FIB-driven isolation of a cryo-lamella from the desired region of a cell or organism.

A view of the FIB/SEM chamber containing showing the in-vacuum section of the PIE-scope. An objective is mounted on a high-precision motorized stage for sample focusing (LM focus drive). The light from the external arm (cyan arrow) is delivered through a glass flange and directed into the objective by a mirror.

ACTIVITY PLAN

  1. Understand the mechanism of acid-induced activation of MPEG-1.
  2. Test the idea that MPEG-1 forms pores in microbes in trans while tethered to the inner leaf of the phagolysosome membrane.
  3. Develop automation techniques for cryo-lamella production to permit in situ structural biology.
  4. Develop software to drive in situ structural biology.

HIGHLIGHT

Scientists make inroads in understanding how immune cells kill bacteria

Gaining knowledge on how our bodies manage to kill bacteria helps scientists learn why one person’s immune system may be working better, worse or differently than others. This is key to ensuring we understand how to fight infections and unlocking the mysteries of our immune systems. The work is published in the journal Nature Communications.

A macrophage is a type of immune cell that is responsible for detecting, engulfing and destroying pathogens, particularly bacteria. By engulfing the bacteria, a macrophage can safely kill the bacteria inside a small sac it creates, known as a phagolysosome. Once this process is complete it then passes debris and information onto other immune cells, kick-starting an immune process that allows our bodies to clear the infection.

Scientists know from previous studies that proteins inside the phagolysosome can create small holes in the membrane, known as a pore, to help destroy the engulfed bacteria. One such protein, MPEG1, which this study focuses on, is in a superfamily of proteins that are known to attack membranes by making pores.

“We knew MPEG1 was found in macrophages, and we also knew that it was related to the membrane attack complex, which targets bacteria. But we didn’t know how the MPEG1 targeted the bacteria inside the macrophage, and we didn’t know how it didn’t damage itself while destroying the bacteria,” said A/Prof. Michelle Dunstone, Associate Investigator from the ARC Imaging Centre of Excellence.

Now, in a world-first, the scientists from the ARC Centre of Excellence in Advanced Molecular Imaging have used cryo-Electron Microscopy (cryo-EM) to discern the first three-dimensional views of MPEG1. Their work demonstrates that MPEG1 is in a state primed and ready to make a pore. Importantly, this exists in macrophages but is inactive until the phagolysosome produces acid (which occurs during the maturation of a phagolysosome). The change in pH activates the pre-pore structure, causing the MPEG1 protein to rearrange themselves into the final pore structure. This impressive change in shape enables pore forming proteins, like MPEG1, to punch holes into bacterial cell membrane surface leading to their death.

Additionally, the study also revealed that MPEG1 seems to tether itself to the macrophage membrane and points the pore-forming machinery away from the macrophage cell. This way it can stay attached to the membrane of the phagolysosome while damaging nearby engulfed bacteria. This appears to be a unique property of this protein and might be another way through which the sac safeguards punching a hole into itself.

“We now know that the immune role of MPEG1 is similar to that of other proteins in this family, but that it only begins to form pores through the process of acidification. This is crucial as it shows us a novel mechanism by which macrophages can control and contain pore-forming proteins like MPEG1 whilst keeping itself safe,” said Prof. James Whisstock, Director of the ARC Imaging Centre of Excellence.

The researchers achieved this by purifying MPEG1 from cells and structurally characterising the molecules using advanced imaging techniques, such as cutting edge cryo-electron microscopy and atomic force microscopy.

“What was fascinating was really a simple question. How does a cell keep itself from harm while harbouring such a dangerous molecular weapon? Cells are delicate, imagine a balloon that has a pin inside it, that it needs for protection, but at the same time it has to ensure that it isn’t harmed by the pin itself. This is the same problem our immune system has had to face in the case of MPEG1. How to kill intracellular bacteria without harming the immune cell itself,” said Mr Charles Bayly-Jones, a PhD Student in Prof. Whisstock’s team.

“This may be able to help us understand why some bacteria might be resistant to destruction by macrophages. This will then give us a better idea of how to keep the immune system functioning, or what may be occurring to prevent it from functioning at its optimal ability,” said Dr Siew Siew Pang, a Postdoctoral Researcher from the ARC Imaging Centre of Excellence.

“In real-world terms, this knowledge can help us understand why MPEG1 mutations would play a role in patients suffering from certain bacteria, such as pulmonary nontuberculous mycobacterial infections,” said Mr Bayly-Jones.

What remains to be shown is whether the MPEG1 structure stays attached to the phagolysosome, while creating pores in the engulfed bacteria. It may detach from the phagolysosome due to some unknown process, before behaving much like other pore-forming proteins would. These are the questions the ARC Scientists may tackle next.

Depicting the proposed mechanism by which MPEG1 acts to kill bacteria within the macrophage phagolysosome. MPEG1 orientation prevents damaging the macrophage membrane.
Credit:
Charles Bayly-Jones

ACHIEVEMENT

Development of new electron microscopy technology increases imaging effectiveness, safety and accessibility

Understanding the biomolecules inside a cell – the way that they interact, how they organise themselves and how they give rise to life – is one of the fundamental goals of cell biology. It can help us understand diseases and disorders and help develop more effective treatments.

One major obstacle for researchers is being able to clearly see macromolecular complexes (groups of biomolecules working together as one structure). Cells are highly active, with lots of moving parts, and in order to focus on a specific region, the cell first has to be stopped via cryofreezing. A cell might also need to be thinned so the electron microscope can penetrate its layers and see the contents within. To overcome these issues scientists will often use electron and light microscopy in correlation to select, image and thin the cellular region of interest.

“It was a labour-intensive process, which required researchers to move the samples across various pieces of equipment and microscopes while keeping the sample at cryo temperatures at all times to avoid the sample being ruined,” said Dr Sergery Gorelick from Monash University’s Biomedicine Discovery Institute and lead author on the paper. “There was too much room for human error, and this is what we wanted to improve.”

Now, scientists from the Australian Research Council (ARC) Imaging Centre have designed an innovative method, which allows all critical steps to be undertaken in one place, decreasing the chances of contamination, and increasing accuracy by enabling precision thinning and instant confirmation that the area of interest hasn’t been lost in the thinning process.

The new design is an integrated cryo-Focused Ion Beam and light microscope setup called the Photon Ion Electron microscope (PIE-scope) that enables direct and rapid isolation of cellular regions.

“Up to now, essentially what we were doing was cutting and imaging blindly,” Monash University author A/Prof. Alex de Marco commented. “The microscope is something that everybody who’s ever done this type of work has been waiting for.”

“This new approach will make cryo-correlative workflow safer and more accessible,” said Prof. James Whisstock, Director of the ARC Imaging Centre. “We designed PIE-scope to enable retrofitting of existing microscopes, which will increase the throughput and accuracy on projects requiring correlative microscopy to target protein complexes.”

The paper, published in eLife Sciences, presents the novel microscope design and software, which allows the targeting of a region in a cryo-preserved cell with a simple workflow and a precision of ~100 nanometers. The PIE-scope is creating much excitement in the field of cell and structural biology, it was downloaded by more than 500 people in the first 24 hours of publication and two companies are looking at commercialising it.

The work was a collaboration of ARC Centre of Excellence in Advanced Molecular Imaging, Monash University’s Biomedicine Discovery Institute, Ramaciotti Centre for Cryo-Electron Microscopy, Monash University’s School of Biological Sciences, Monash University’s Department of Anatomy and Developmental Biology, Biomedicine Discovery Institute, University of Warwick, EMBL Australia.

Top: Dr Sergey Gorelick and A/Prof. Alex de Marco in front of the PIE-scope.z

Credit: Monash Biomedicine Discovery Institute

Bottom: ROI identification, targeting and cryo-lamella preparation in C. elegans larvae, (g, k) show SEM and optical images, respectively, of the intermediate steps in the lamella, thinning.