The Price of Tags in Protein Localization Studies

Margolin, William

doi:10.1128/jb.01640-12

Cited by 71 publications

(72 citation statements)

References 35 publications

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“…The development of less-bulky fluorescent tags and membrane-permeable organic dyes for protein visualization in bacteria (Griffin et al, 1998;Charbon et al, 2011), combined with near-native or adjustable expression levels, should ensure more physiologically relevant observations of subcellular distributions in the future. Currently, artefactual localization patterns -often polar accumulations -remain a concern as they can arise upon overexpression, misfolding or fusion to some fluorescent tags, depending on the protein being visualized (Winkler et al, 2010;Landgraf et al, 2012;Margolin, 2012;Swulius and Jensen, 2012). In addition, the quantification of subcellular patterns should become an integral part of any localization study.…”

Section: Discussionmentioning

confidence: 99%

How do bacteria localize proteins to the cell pole?

Laloux

Jacobs‐Wagner

2014

Journal of Cell Science

163

158

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It is now well appreciated that bacterial cells are highly organized, which is far from the initial concept that they are merely bags of randomly distributed macromolecules and chemicals. Central to their spatial organization is the precise positioning of certain proteins in subcellular domains of the cell. In particular, the cell poles -the ends of rod-shaped cells -constitute important platforms for cellular regulation that underlie processes as essential as cell cycle progression, cellular differentiation, virulence, chemotaxis and growth of appendages. Thus, understanding how the polar localization of specific proteins is achieved and regulated is a crucial question in bacterial cell biology. Often, polarly localized proteins are recruited to the poles through their interaction with other proteins or protein complexes that were already located there, in a so-called diffusion-and-capture mechanism. Bacteria are also starting to reveal their secrets on how the initial pole 'recognition' can occur and how this event can be regulated to generate dynamic, reproducible patterns in time (for example, during the cell cycle) and space (for example, at a specific cell pole). Here, we review the major mechanisms that have been described in the literature, with an emphasis on the self-organizing principles. We also present regulation strategies adopted by bacterial cells to obtain complex spatiotemporal patterns of protein localization.

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Section: Discussionmentioning

confidence: 99%

How do bacteria localize proteins to the cell pole?

Laloux

Jacobs‐Wagner

2014

Journal of Cell Science

163

158

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“…Additionally, a putative caveat of tagging strategies is altered protein behavior like mislocalization, changes in protein stability, or a dominant negative regulatory effect (Margolin 2012).…”

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confidence: 99%

The developmental proteome of Drosophila melanogaster

et al. 2017

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Drosophila melanogaster is a widely used genetic model organism in developmental biology. While this model organism has been intensively studied at the RNA level, a comprehensive proteomic study covering the complete life cycle is still missing. Here, we apply label-free quantitative proteomics to explore proteome remodeling across Drosophila’s life cycle, resulting in 7952 proteins, and provide a high temporal-resolved embryogenesis proteome of 5458 proteins. Our proteome data enabled us to monitor isoform-specific expression of 34 genes during development, to identify the pseudogene Cyp9f3Ψ as a protein-coding gene, and to obtain evidence of 268 small proteins. Moreover, the comparison with available transcriptomic data uncovered examples of poor correlation between mRNA and protein, underscoring the importance of proteomics to study developmental progression. Data integration of our embryogenesis proteome with tissue-specific data revealed spatial and temporal information for further functional studies of yet uncharacterized proteins. Overall, our high resolution proteomes provide a powerful resource and can be explored in detail in our interactive web interface.

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“…Or is helical localization perhaps due to the effect of fluorescent fusions on tagged proteins? It is now becoming clear that different imaging techniques are required to corroborate the observed localization patterns (41). Cryo-electron tomography corroborated the absence of long (Ͼ80-nm) MreB filaments near or along the inner membrane of six different rod-shaped bacteria, although use of correlative cryo-light and electron tomography of GFP-MreB allowed the identification of cytoplasmic MreB bundles, showing that MreB is indeed detectable by cryo-ET (42).…”

Section: Mreb and Mreb-like Proteinsmentioning

confidence: 97%

Multidimensional View of the Bacterial Cytoskeleton

et al. 2013

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bThe perspective of the cytoskeleton as a feature unique to eukaryotic organisms was overturned when homologs of the eukaryotic cytoskeletal elements were identified in prokaryotes and implicated in major cell functions, including growth, morphogenesis, cell division, DNA partitioning, and cell motility. FtsZ and MreB were the first identified homologs of tubulin and actin, respectively, followed by the discovery of crescentin as an intermediate filament-like protein. In addition, new elements were identified which have no apparent eukaryotic counterparts, such as the deviant Walker A-type ATPases, bactofilins, and several novel elements recently identified in streptomycetes, highlighting the unsuspected complexity of cytostructural components in bacteria. In vivo multidimensional fluorescence microscopy has demonstrated the dynamics of the bacterial intracellular world, and yet we are only starting to understand the role of cytoskeletal elements. Elucidating structure-function relationships remains challenging, because core cytoskeletal protein motifs show remarkable plasticity, with one element often performing various functions and one function being performed by several types of elements. Structural imaging techniques, such as cryo-electron tomography in combination with advanced light microscopy, are providing the missing links and enabling scientists to answer many outstanding questions regarding prokaryotic cellular architecture. Here we review the recent advances made toward understanding the different roles of cytoskeletal proteins in bacteria, with particular emphasis on modern imaging approaches.

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The Price of Tags in Protein Localization Studies

Cited by 71 publications

References 35 publications

How do bacteria localize proteins to the cell pole?

How do bacteria localize proteins to the cell pole?

The developmental proteome of Drosophila melanogaster

Multidimensional View of the Bacterial Cytoskeleton

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