Principles of cell and tissue organization


Endocytosis and disease

Quantitative analysis of mycobacteria-host interactions in cells and tissues

Mycobacterium tuberculosis is often considered the most successful bacterial pathogen due to its very high level of penetrance in the human population. The bacteria owes a large part of its remarkable success to its ability to manipulate various host cellular processes to suit its requirements.

In our lab we are interested in the cell biology of M. tuberculosis infection at the cellular and tissue levels. Towards this, we are exploring the use of quantitative image analysis in the study of host-mycobacteria interactions. We have recently established tools to quantitatively evaluate the trafficking and survival of mycobacteria within infected human primary macrophages and have identified several compounds that kill intracellular bacteria by modulating the host cellular processes. We have further developed methods to deconvolve the intracellular effects of these chemicals and identified the cellular processes they affect (Sundaramurthy et al, Cell Host and Microbes, 2013). Currently we are focusing on two different projects.

  • We are members of the PreDiCT-TB consortium where we work in close collaboration with industry partners to use the quantitative intracellular mycobacterial survival assay to evaluate new and novel TB drugs and combinations.
  • In collaboration with the David Russell lab (Cornell University), we are applying quantitative image analysis tools to study the organization of M. tuberculosis infection in tissue. In this project, we are developing quantitative image analysis in 3D.
Fig. 2. Validation of Three Selected Compounds for Intracellular Mycobacterial Survival

(A–D) Representative images of infected cells treated with DMSO (A), Nortriptyline (B), prochlorperazine edisylate (PE) (C), and Haloperidol (D) at a concentration of 10 mM for 48 hr after infection. Arrowheads indicate GFP-positive structures that are identified as ‘‘bacteria’’ by the QMPIA. Scale bar, 10 mm (from Sundaramurthy et al., 2013)

Systems biology study of factors leading to perturbed endocytosis in Huntington's Disease

This project, funded by the CHDI foundation, is focusing on Huntington’s Disease (HD), a neurodegenerative disorder caused by mutant Huntingtin (mHtt) that impairs cognitive capacity and causes motor deficits along with involuntary movements. We aim at understanding how endocytosis is perturbed in HD and how membrane trafficking defects contribute to the overall pathogenesis.

To this end, we apply the Multiparametric Quantitative Image Analysis (QMPIA) platform that we developed in the past years to analyze the phenotypic signatures of perturbed vesicle motility and marker distribution in HD cells. In parallel, we have established a Systems Biology pipeline to predict and validate candidate interactors of mHtt as well as small compounds suppressing membrane trafficking defects in HD.

By combining the technology of the QMPIA platform with small compound and RNAi screens, we identify gene and compound candidates that either enhance or antagonize the mHtt activity and can cluster them into functional modules. Our ultimate goal is to validate and further improve the predictive power of our phenotypic System Biology pipeline for the dissection of molecular pathogenesis and drug discovery as opposed to traditional target-based approaches.

Fig. 1

On the left: maximum intensity projection of motile GFP-Rab5-labeled early endosomes (from live movies). Early endosomes move progressively over long distances in wild type (WT) cells (top left) as opposed to the virtual arrest of these organelles in Huntington's Disease (HD) cells (bottom left). On the right: the defect in early endosome motility (left images) causes perturbed cargo sorting and routing in HD. Cells were incubated with fluorescently labeled transferrin over 10 min to stain endocytic compartments, fixed and imaged. Note the even distribution of cargo in WT cells (top right) as opposed to the striking cargo accumulation in the cell periphery in HD cells (bottom right, yellow arrowheads). Such trafficking defects have a detrimental impact on neurotrophic support in the CNS of HD patients, thereby contributing to neurodegeneration.

Zerial lab