Objective Clustering of Proteins Based on Subcellular Location Patterns

The goal of proteomics is the complete characterization of all proteins. Efforts to characterize subcellular location have been limited to assigning proteins to general categories of organelles. We have previously designed numerical features to describe location patterns in microscope images and developed automated classifiers that distinguish major subcellular patterns with high accuracy (including patterns not distinguishable by visual examination). The results suggest the feasibility of automatically determining which proteins share a single location pattern in a given cell type. We describe an automated method that selects the best feature set to describe images for a given collection of proteins and constructs an effective partitioning of the proteins by location. An example for a limited protein set is presented. As additional data become available, this approach can produce for the first time an objective systematics for protein location and provide an important starting point for discovering sequence motifs that determine localization

Characterizing an Alternative Chloroplast Outer Membrane Targeting Signal in Arabidopsis thaliana

Chloroplasts are organelles that are unique to plant and algal cells and are the site of photosynthesis. Though chloroplasts contain their own genome, an estimated 95% of chloroplast proteins are encoded in the nucleus, and therefore rely on post-translational targeting to the organelle. The majority of known chloroplast proteins are targeted to the chloroplast interior by cleavable signals at the N-terminal end of preproteins known as transit peptides. The translocon at the outer envelope membrane of chloroplasts (Toc) is a multimeric complex that recognizes and binds N-terminal transit peptides at the cytosolic surface of chloroplasts. Though transit peptides are necessary and sufficient for guiding nuclear-encoded preproteins into the chloroplast interior, the nature of sequence information of transit peptides is not fully understood due to their high divergence in length and composition. Over the last nine years, the number of proteins known or predicted to reside in the chloroplast outer envelope membrane of Arabidopsis has tripled to one hundred and seventeen. Although the functions for some of these outer envelope proteins (OEPs) have been characterized, the precise mechanism of their targeting to the chloroplast outer membrane has not been fully elucidated. Besides Toc75, the targeting mechanisms used by OEPs that have been characterized do not involve an N-terminal transit peptide. The bioinformatics tool ChloroP can be used to predict if amino acid sequences contain an N-terminal transit peptide. Recently, ChloroP analysis and protoplast transient expression assays were used to identify a novel chloroplast targeting signal in the C-terminus of the chloroplast preprotein receptor Toc159 in Bienertia sinuspersici (Lung and Chuong, 2012). Toc159 was also shown to lack a canonical transmembrane domain typically present in OEPs. While the unique C-terminal targeting sequence has been partially characterized in Toc159 (Lung et al., 2014), it left open the question of whether this type of signal is unique to Toc159, or if it is used by other OEPs as well. In the current study, to determine if other OEPs use this novel targeting pathway, ChloroP analysis identified eight potential candidates possessing the putative C-terminal targeting signal in Arabidopsis. Transient protoplast expression assays have been performed on OEP18, the protein predicted with the highest ChloroP score, to determine its subcellular localization and the sequences required for its targeting to chloroplasts. The primary purpose of the current study was to establish whether chloroplast outer membrane proteins other than Toc159 use a similar C-terminal targeting signal. Overall, the data in this thesis suggest that some OEPs other than Toc159, such as OEP18, may use this novel targeting pathway

Subcellular protein expression models for microsatellite instability in colorectal adenocarcinoma tissue images

Background New bioimaging techniques capable of visualising the co-location of numerous proteins within individual cells have been proposed to study tumour heterogeneity of neighbouring cells within the same tissue specimen. These techniques have highlighted the need to better understand the interplay between proteins in terms of their colocalisation. Results We recently proposed a cellular-level model of the healthy and cancerous colonic crypt microenvironments. Here, we extend the model to include detailed models of protein expression to generate synthetic multiplex fluorescence data. As a first step, we present models for various cell organelles learned from real immunofluorescence data from the Human Protein Atlas. Comparison between the distribution of various features obtained from the real and synthetic organelles has shown very good agreement. This has included both features that have been used as part of the model input and ones that have not been explicitly considered. We then develop models for six proteins which are important colorectal cancer biomarkers and are associated with microsatellite instability, namely MLH1, PMS2, MSH2, MSH6, P53 and PTEN. The protein models include their complex expression patterns and which cell phenotypes express them. The models have been validated by comparing distributions of real and synthesised parameters and by application of frameworks for analysing multiplex immunofluorescence image data. Conclusions The six proteins have been chosen as a case study to illustrate how the model can be used to generate synthetic multiplex immunofluorescence data. Further proteins could be included within the model in a similar manner to enable the study of a larger set of proteins of interest and their interactions. To the best of our knowledge, this is the first model for expression of multiple proteins in anatomically intact tissue, rather than within cells in culture.QNRF grant NPRP 5-1345-1-228. BBSRC and University of Warwick Institute of Advanced Study

Quantifying the distribution of probes between subcellular locations using unsupervised pattern unmixing

Motivation: Proteins exhibit complex subcellular distributions, which may include localizing in more than one organelle and varying in location depending on the cell physiology. Estimating the amount of protein distributed in each subcellular location is essential for quantitative understanding and modeling of protein dynamics and how they affect cell behaviors. We have previously described automated methods using fluorescent microscope images to determine the fractions of protein fluorescence in various subcellular locations when the basic locations in which a protein can be present are known. As this set of basic locations may be unknown (especially for studies on a proteome-wide scale), we here describe unsupervised methods to identify the fundamental patterns from images of mixed patterns and estimate the fractional composition of them

Organic Cation Transporter 3 (OCT3) Is Localized to Intracellular and Surface Membranes in Select Glial and Neuronal Cells Within the Basolateral Amygdaloid Complex of Both Rats and Mice

Organic cation transporter 3 (OCT3) is a high-capacity, low-affinity transporter that mediates corticosterone-sensitive uptake of monoamines including norepinephrine, epinephrine, dopamine, histamine and serotonin. OCT3 is expressed widely throughout the amygdaloid complex and other brain regions where monoamines are key regulators of emotional behaviors affected by stress. However, assessing the contribution of OCT3 to the regulation of monoaminergic neurotransmission and monoamine-dependent regulation of behavior requires fundamental information about the subcellular distribution of OCT3 expression. We used immunofluorescence and immuno-electron microscopy to examine the cellular and subcellular distribution of the transporter in the basolateral amygdaloid complex of the rat and mouse brain. OCT3-immunoreactivity was observed in both glial and neuronal perikarya in both rat and mouse amygdala. Electron microscopic immunolabeling revealed plasma membrane-associated OCT3 immunoreactivity on axonal, dendritic, and astrocytic processes adjacent to a variety of synapses, as well as on neuronal somata. In addition to plasma membrane sites, OCT3 immunolabeling was also observed associated with neuronal and glial endomembranes, including Golgi, mitochondrial and nuclear membranes. Particularly prominent labeling of the outer nuclear membrane was observed in neuronal, astrocytic, microglial and endothelial perikarya. The localization of OCT3 to neuronal and glial plasma membranes adjacent to synaptic sites is consistent with an important role for this transporter in regulating the amplitude, duration, and physical spread of released monoamines, while its localization to mitochondrial and outer nuclear membranes suggests previously undescribed roles for the transporter in the intracellular disposition of monoamines