Now I see you: the imaging toolbox that improves the detection of primary ciliary dyskinesia


Ciliary motility is essential for moving fluids and particles over epithelial surfaces. Frequently, the limitations of quantitative tests and the lack of awareness delay the diagnosis of cilia diseases. In this blog post, we present antibodies- and 3D imaging-based quantitative super-resolution toolbox for the diagnosis of primary ciliary dyskinesia, a disorder of motile cilia. The researchers took part in our Explorer Program. Read on to discover what they found.

Cilia are slender, microscopic, highly conserved hair-like structures or organelles that line the surfaces of individual cells of nearly all mammalian cells. Cilia beat in rhythmic waves and use sweeping motions, moving liquids along internal epithelial tissue in animals. Recent discoveries have revealed that cilia have crucial roles in the signal transduction pathways that regulate intracellular calcium levels and maintain cellular homeostasis. The dysfunction of cilia causes diseases known as ciliopathies.

 

Motile and non‐motile cilia

There are two types of cilia: motile and non-motile cilia. The non-motile cilia (also called primary cilia) typically serve as sensory organelles. Whereas primary cilia have relatively little additional structure, motile cilia have both a central doublet of microtubules as well as inner and outer dynein arms and radial spokes, which are all needed for motility.

There is a third type of cilium, known as nodal cilia, but this is only transiently present in the early embryo and is crucial in the establishment of the left-to right body asymmetry.

Liu-et-al-blog-atlasantibodies-fig1

Figure. 1. Illustration of the motile cilia structure. A basal body and an axoneme composed the cilia. The cilia axoneme is the core and the most prominent structural component of the cilia. TZ, transition zone; DA, distal appendage; BF, basal foot (Liu Z. et al, 2020).

 

 

Motile' cilia are found in the lungs, respiratory tract, and middle ear. Airway clearance of pathogens and particulates relies on motile cilia. These cilia have a rhythmic waving or beating motion. For instance, they work to keep the airways clear of mucus and dirt, allowing us to breathe easily and without irritation. Impaired cilia motility is associated with recurrent infections, leading to a reduction in lung function (bronchiectasis), lung transplant, or death in some cases.

Non-motile cilia are found in the eye, inside the light-sensitive cells (photoreceptors) of the retina and in most cells of the body that are not dividing (with few exceptions). These cilia act like microscopic train-tracks and allow the transport of vital molecules from one end of the photoreceptor to the other. In the kidney, non-motile cilia bend with urine flow and send a signal to alert the cells that there is a flow of urine.

 

Ciliopathies

Ciliopathies are inherited human disorders that affect primary cilia genes. They are caused by both motile and non‐motile cilia dysfunction and are becoming a rapidly expanding disease category. The definition of ciliopathies is evolving while we understand more about them.

At least 35 different ciliopathies, collectively affect nearly all organ systems, with prevalent phenotypes including polycystic kidney disease, retinal degeneration, obesity, skeletal malformations, and brain anomalies.

The genetic heterogeneity, together with the high phenotypic variability, overlapping phenotypes, and the progressive onset of many features during childhood and adolescence, complicates both the clinical and molecular diagnosis of ciliopathies.

Genetic tools such as next-generation sequencing, high-throughput technologies, and advanced bioinformatics tools developed in the last decade are a powerful way of improving our understanding of the biological basis of ciliopathy disorders.

 

FIG2-liu-et-al-atlasantibodies-blog-

Figure 2. Schematic showing rotational polarity in cells from healthy individuals and patients with PCD (Liu et al 2020).

 

A need for quantitative methods for improving PCD diagnosis

Primary ciliary dyskinesia (PCD) is an autosomal recessive genetic condition in which the motile cilia in the respiratory system are defective. PCD affects how cilia grow and function (Figure 2).

Disease caused by proteins functioning in motile cilia can cause different phenotypes such as infertility, cardiac malformation, situs inversus, and lung disease. PCD refers mostly to the lung phenotype.

PCD is a disorder characterized by chronic respiratory tract infections and abnormally positioned internal organs. Faulty genes cause the cilia to be immotile, stiff, uncoordinated, and sometimes in the wrong size or shape. Sometimes the cilia are missing altogether. If the cilia don't work well, bacteria stay in the airways, causing breathing problems, infections, and other disorders.

 

The challenge: from a clinical to a molecular diagnosis

The clinical diagnosis of PCD symptoms like cough and rhinitis is not specific enough for an accurate diagnosis. For this reason, besides the clinical diagnosis, a precise quantitative molecular determination is essential for identifying clear targets for future therapies or for initiating treatments that can stabilize lung function.

However, to date, the molecular diagnosis of PCD has mainly relied on non-quantitative methods with limited sensitivity or that require a priori knowledge of the genes involved.

 

The generation of the PCD proteome map

Over 50 proteins regulating cilia motility are linked to PCD, and more are awaiting validation. To improve the diagnosis of PCD, Liu, and colleagues from the University of Toronto, Canada, generated a PCD imaging proteome map and developed a toolbox of quantitative imaging methods.

The study, published in Science Translational Medicine, 2020:12(535), uses a combination of imaging approaches that produce quantitative results useful to improve upon current, non-quantitative diagnostic methods.

The new super-resolution based imaging methods comprise antibodies immunoassays combined with 3D structured illumination microscopy (3D-SIM) and stochastic optical reconstruction microscopy (STORM). Super-resolution microscopy on nasal airway cells from PCD patients can identify proteins in the wrong locations, defects in cellular structure, and impaired beating movements in the cilia. 

The goals of this quantitative super-resolution microscopy workflow are:

  • to increase sensitivity and throughput.
  • to detect structural defects in PCD patients’ cells.
  • to quantify motility defects caused by yet to be found PCD genes.

 

Now I see you, cilia!

A panel of 21 commercially available antibodies directed against PCD proteins was validated to detect molecular defects in multiciliated airway cells from patients with PCD. Ciliated
 epithelium
 was
 obtained 
by
 nasal
 brush
 biopsy.

The table below lists some antibodies from Atlas Antibodies used in the study.

Product name Catalogue Nr Product name Catalogue Nr
Anti-ARMC4  HPA037829 Anti-GAS2L2 HPA044370
Anti-CCDC39 HPA035364 Anti-GAS8 HPA041311
Anti-CCDC40 HPA022974 Anti-HYDIN HPA067155
Anti-CCDC65 HPA038520 Anti-LRRC6 HPA028058
Anti-CCDC114 HPA042524 Anti-OFD1 HPA031104
Anti-CCDC151 HPA044184 Anti-PIH1D3 HPA072496
Anti-C21ORF59 HPA028849 Anti-RPGR HPA001593
Anti-DNAAF1 HPA041166 Anti-RSPH4A HPA031196
Anti-DNAAF2 HPA004113 Anti-RSPH9 HPA031703
Anti-DNAH5 HPA037470 Anti-SPAG1 HPA023748
Anti-DNAH11 HPA056474 Anti-SPAG6 HPA038440
Anti-DNAI1 HPA021649 Anti-SPEF2 HPA039606
Anti-DNALI1 HPA028305 Anti-TTC25 HPA023908
Anti-DNAL1 HPA053129    
Anti-DNAJB13 HPA052465    
Anti-DNAH9 HPA052641    

 

After immunostaining, the isolated multiciliated cells were imaged by 3D-SIM or STORM, a technique able to achieve ~25-nm in-plane and ~50-nm axial resolution, and which allows detailed visualization of distributions of specific PCD proteins thus increasing the final image resolution (Figure 3).

liu-et-al-antibody-staining-atlasantibodies-fig3

Figure 3. Comparison between STORM imaging and conventional fluorescence imaging. STORM imaging of GAS8 with the anti-GAS8 polyclonal antibody (HPA041311) is visible in green. Insets in white boxes are high-magnification images of three ciliary regions showing periodic distribution. Box on the far-right shows high-magnification image of an intact cilium from the STORM image on the left (Liu et al 2020).

 

Learning Points

  • Primary ciliary dyskinesia (PCD) is a genetic condition in which cilia in the respiratory system are malfunctioning.
  • The diagnosis of PCD is frequently delayed until well after bronchiectasis has developed. 
  • PCD diagnosis relies on nonquantitative methods with limited sensitivity.
  • The new super-resolution imaging method published by Liu et al., 2020 comprises antibodies immunoassays combined with structured illumination microscopy (3D-SIM) and stochastic optical reconstruction microscopy (STORM).
  • 3D-SIM/STORM is a sensitive, simple, and powerful tool for improving the diagnosis of motile ciliopathies and can integrate or replace some current clinical diagnostic methods.
  • Together, this data show that super-resolution methods are powerful tools for improving diagnosis of motile ciliopathies.

 

 

Reference

Zhen Liu, Quynh P. H. Nguyen, Qingxu Guan, Alexandra Albulescu, Lauren Erdman, Yasaman Mahdaviyeh, Jasmine Kang, Hong Ouyang, Richard G. Hegele, Theo Moraes, Anna Goldenberg, Sharon D. Dell, Vito Mennella.

A quantitative super-resolution imaging toolbox for diagnosis of motile ciliopathies. 

Science Translational Medicine, 2020:12(535)

doi:10.1126/scitranslmed.aay0071

Topics:

biomarkers / ciliopathies / imaging / atlasantibodies

Written by Dr. Laura Pozzi

Dr. Laura Pozzi is a scientific writer at Atlas Antibodies. She holds a Ph.D. in Life and Biomolecular Science from the Open University of London in collaboration with the Mario Negri Institute for Pharmacological Research in Milan. Laura has worked as a researcher at Karolinska Institutet in Sweden and more recently as associated editor. She has a long track record of scientific publications as a first author and as coauthor. Her research focus on neuroscience with a broad experience in antibodies validation and immunohistochemistry techniques.

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