THEME 3 MOLECULAR IMAGING OF T-CELL ACTIVATION

A new unbiased analysis method based on point pattern statistics identified nine different spatial organisation of the T cell receptor in a human T lymphocyte (also called a T cell).

“With our new single molecule microscope, we can now measure distances between molecules in intact immune cells on the biologically relevant scale.”

More than 30 years after the discovery of the T cell receptor, we still do not know how signalling begins. It is important that we work out how antigen binding initiates intracellular signalling because these signals shape the resulting immune response. With new super-resolution fluorescence microscopy and single molecule imaging approaches, we aim to map the molecular level while retaining the spatial organisation of intact cells. We are developing new instruments, new analysis methods and new molecular biology tools in an interdisciplinary research program that spans physics, chemistry, mathematics and biology.

PROF. KATHARINA (KAT) GAUS

AT A GLANCE

Super-resolution fluorescence microscopy promises the opportunity to place single molecules and multi-molecular complexes in the cellular context and in turn understand how molecular organisation of cells lead to cellular outcomes. A key question in cellular immunology is how antigen recognition leads to intracellular signalling on which T cell fate decisions are based. The T cell signalling field is stymied as we do not understand how engagement of the T cell receptor (TCR) on the extracellular side initiates phosphorylation of the constitutively associated CD3 dimers on the intracellular side. It is not known how diverse signalling outcomes are encoded by a common TCR-CD3 signalling transduction process. We hypothesise that the spatial organisations are key to signal initiation, integration of signals from multiple receptor, and the regulation of a highly plastic signalling network.

HIGHLIGHT

3D-printed microscope makes sensitive disease diagnosis more accessible

A 3D-printed microscope created by Imaging CoE scientists at UNSW Medicine has the potential to make rapid disease screening and diagnosis simpler – and it’s free for anyone to download and use.

The researchers at Single Molecule Science have shared the full 3D printing instructions, analysis and optical design details in a paper in open access journal Nature Communications. Dr Emma Sierecki co-led the team.

“Our intention is that researchers who have never done single-molecule fluorescence detection before can download the files, print the scaffolding, put the three little optical elements in, and start working with the microscope. The optical elements are pre-aligned,” said Dr Yann Gambin, EMBL Australia Group Leader at UNSW Medicine’s Single Molecule Science.

The compact plug-and-play microscope – called AttoBright – has the power to detect molecules associated with diseases like Parkinson’s disease and tuberculosis. For a mere fraction of the cost of a traditional instrument, any research laboratory can tap into its superior sensitivity, as can researchers who need to take it out in the field and other resource poor settings.

The cost saving – while substantial – isn’t AttoBright’s only advantage: it is simple to use and does not require the specialist training that traditional confocal microscopes need.

“Instead of training people for weeks or a month on single-molecule acquisition, they can start using the instrument in five minutes,” said Dr Gambin.

To demonstrate the sensitivity and accuracy of their single-molecule sensor, the team used it to detect alpha-synuclein. The clumping of this protein is linked with Parkinson’s disease.

“We show that this simple instrument is more than 100,000 times more sensitive, compared with the plate readers that researchers typically use for this screening,” Dr Gambin said.

Article source: UNSW Newsroom 

AttoBright is a 3D-printed microscope.

ACTIVITY PLAN

  1. Identify subunits and subunit arrangements of protein complexes in intact cells, exploiting the photophyiscal properties of fluorophores for molecular counting.
  2. Link protein localisation and spatial organisations to function, distinguishing signalling from non-signalling molecules and determine the environmental factors.
  3. Combining biochemical assays with single molecule imaging to establish a conceptual and theoretical framework of signal integration at the receptor level.
  4. Map signalling networks by developing new statistical analysis for single molecule data that reveals the information flow in intracellular signalling networks.
  5. Disseminating microscopy hardware and software. To make a lasting impact on the scientific community and connect with
    end-users, we are not just developing novel microscope hardware and software but also developing avenues to cost efficiently replicate and disseminate our approaches. For example, we are exploring how 3-D printing could aid in the prototyping of
    hardware and collaborating with MASSIVE to develop approaches for handling and processing large imaging-based data.

ACHIEVEMENT

Tinkering with toy cars to improve immune cell signalling

To understand precisely how the T cell receptor operates to drive an immune response – or decide to remain at rest – Associate Investigator, Dr Jesse Goyette is examining individual components of the T cell receptor to decipher their roles in T cell receptor signalling.

T cell receptors re-engineered by chopping and changing different parts of the signalling machinery are reintroduced into T cells, and Dr Goyette uses super-resolution microscopy techniques to observe how these T cells respond to antigen. Knowing what makes T cell receptors more sensitive, or work more efficiently, means the right adjustments can be made to improve signalling properties and enhance immune responses.

Modified immune receptors have already found utility in the clinic. Chimeric antigen receptors, or CARs, are immune receptors modified to specifically detect and destroy cancer cells and other targets. A new form of immunotherapy using CARs appears to be effective in treating B cell lymphomas and other cancers.

“This is a way of redirecting the cell’s signalling machinery to a target that you want using a chimeric receptor that you could design and build,” says Dr Goyette, who now jointly leads the Lymphocyte Signalling group with Scientia Professor Katharina Gaus, Chief Investigator at the Imaging CoE.

“The T cell receptor complex is quite modular, it seems really robust,” said Dr Goyette.

Inspired by the CAR system, Jesse, with his team at Single Molecule Science, is making modifications to other parts of the T cell receptor complex – inside the cells – to investigate the signalling machinery that initiates and transmits signals that make the decision to either switch T cells on, or not.

“As an experimental system, we can isolate different parts of the T cell receptor that may be important for signalling. I like to call them toy CARs,” Dr Goyette said.

By tinkering with these toy CARs, researchers can learn how to make T cells more efficient.

Article Source: UNSW Newsroom

Microscopy image of T cells bearing chimeric antigen receptors; negative regulator, blue, a recruited signalling kinase, yellow.
Dr Jesse Goyette
Dr Jesse Goyette

Interactive Science Expo, UNSW 

In November, the research students and staff at Single Molecule Science (SMS) from the Imaging CoE’s UNSW Node, hosted an Interactive Science Expo to engage audiences with multisensory exhibits. Special guest, David Choi, a deafblind artist from New Zealand, shared his story and his ‘Brain Tree’ artwork that depicted the five senses.

Staff and students of SMS used tactile displays and hands-on activities to communicate Imaging CoE and SMS’s research. To take audiences with vision impairment through a typical experiment in the laboratory, one team recorded the sounds produced by equipment and reproduced some of the smells a researcher would encounter at different steps of isolating and analysing proteins.

Another team created a replica of the cell culture system they use to study how cells sense stretching and vibration—like the hair cells in human ears. When touched, flashing LED lights on the interactive model demonstrated how electrical messages are transmitted through a cell when it is stretched.

The interactive science expo was attended by over 100 visitors including, clients and affiliates of Guide Dogs NSW/ACT and Vision Australia, a Deaf Learners group, student groups from the Royal Institute for Deaf and Blind Children, and hearing support units from schools around Sydney. To bridge the accessibility gap for deaf attendees, interpreters from the Deaf Society played a vital role and their rapid response communications skills were impressive.

The success of the event was clear from the response of the audience, who were engaged, excited, appreciative and very curious. Teachers were also eager to explore the materials and activities, and keen to incorporate some of the ideas into their science classrooms.

The expo was sponsored by the UNSW Division of Equity Diversity and Inclusion; Imaging CoE; and the UNSW School of Medical Sciences.

 

Dr Jesse Goyette with some guests at the 2019 Interactive Science Expo