Systematic genetics and single-cell imaging reveal widespread morphological pleiotropy and cell-to-cell variability.

Mojca Mattiazzi Usaj,Ermira Shuteriqi,Carles Pons,Helena Friesen,Nil Sahin,Brenda J Andrews,Patrick Aloy,Matej Usaj,Charles Boone,Quaid Morris,Myra Paz D Masinas,Aleksei Shkurin

doi:10.15252/msb.20199243

Abstract

Our ability to understand the genotype‐to‐phenotype relationship is hindered by the lack of detailed understanding of phenotypes at a single‐cell level. To systematically assess cell‐to‐cell phenotypic variability, we combined automated yeast genetics, high‐content screening and neural network‐based image analysis of single cells, focussing on genes that influence the architecture of four subcellular compartments of the endocytic pathway as a model system. Our unbiased assessment of the morphology of these compartments—endocytic patch, actin patch, late endosome and vacuole—identified 17 distinct mutant phenotypes associated with ~1,600 genes (~30% of all yeast genes). Approximately half of these mutants exhibited multiple phenotypes, highlighting the extent of morphological pleiotropy. Quantitative analysis also revealed that incomplete penetrance was prevalent, with the majority of mutants exhibiting substantial variability in phenotype at the single‐cell level. Our single‐cell analysis enabled exploration of factors that contribute to incomplete penetrance and cellular heterogeneity, including replicative age, organelle inheritance and response to stress.

Highlights

Our ability to understand the genotype-to-phenotype relationship is hindered by the lack of detailed understanding of phenotypes at a single-cell level
To enable a quantitative analysis of subcellular compartment morphology, we developed a high-throughput (HTP) image-based pipeline coupled to single-cell image analysis (Fig 1A)
We focused on (i) SLA1, encoding an endocytic adaptor protein, marking the coat complex associated with early endocytic sites at the plasma membrane; (ii) SAC6, encoding yeast fimbrin, marking actin patches that are required for early endocytosis events; (iii) a late endosomal marker, SNF7, encoding a subunit of the ESCRT-III complex involved in the sorting of transmembrane proteins into the multivesicular body (MVB) pathway; and (iv) a marker for the vacuolar membrane, VPH1, encoding subunit “a” of the vacuolar ATPase (V-ATPase) VO domain (Fig 1B)

Summary

Introduction

Our ability to understand the genotype-to-phenotype relationship is hindered by the lack of detailed understanding of phenotypes at a single-cell level. Our unbiased assessment of the morphology of these compartments—endocytic patch, actin patch, late endosome and vacuole— identified 17 distinct mutant phenotypes associated with ~1,600 genes (~30% of all yeast genes). Half of these mutants exhibited multiple phenotypes, highlighting the extent of morphological pleiotropy. Quantitative analysis revealed that incomplete penetrance was prevalent, with the majority of mutants exhibiting substantial variability in phenotype at the single-cell level. Our single-cell analysis enabled exploration of factors that contribute to incomplete penetrance and cellular heterogeneity, including replicative age, organelle inheritance and response to stress

Methods

Results

Conclusion