McKinley Lab Publications
All Publications
2023
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2023. Mechanisms of Regeneration and Fibrosis in the Endometrium. Annual review of cell and developmental biology. 39:197-221. Pubmed: 37843929 DOI:10.1146/annurev-cellbio-011723-021442 Ang CJ, Skokan TD, McKinley KL. 2023. Mechanisms of Regeneration and Fibrosis in the Endometrium. Annual review of cell and developmental biology. 39:197-221. Pubmed: 37843929 DOI:10.1146/annurev-cellbio-011723-021442 The uterine lining (endometrium) regenerates repeatedly over the life span as part of its normal physiology. Substantial portions of the endometrium are shed during childbirth (parturition) and, in some species, menstruation, but the tissue is rapidly rebuilt without scarring, rendering it a powerful model of regeneration in mammals. Nonetheless, following some assaults, including medical procedures and infections, the endometrium fails to regenerate and instead forms scars that may interfere with normal endometrial function and contribute to infertility. Thus, the endometrium provides an exceptional platform to answer a central question of regenerative medicine: Why do some systems regenerate while others scar? Here, we review our current understanding of diverse endometrial disruption events in humans, nonhuman primates, and rodents, and the associated mechanisms of regenerative success and failure. Elucidating the determinants of these disparate repair processes promises insights into fundamental mechanisms of mammalian regeneration with substantial implications for reproductive health. -
Zwick RK, Kasparek P, Palikuqi B, Viragova S, Weichselbaum L, McGinnis CS, McKinley KL, Rathnayake A, Vaka D, Nguyen V, Trentesaux C, Reyes E, Gupta AR, Gartner ZJ, Locksley RM, Gardner JM, Itzkovitz S, Boffelli D, Klein OD. 2023. Epithelial zonation along the mouse and human small intestine defines five discrete metabolic domains. bioRxiv : the preprint server for biology. Pubmed: 37790430 DOI:10.1101/2023.09.20.558726 Zwick RK, Kasparek P, Palikuqi B, Viragova S, Weichselbaum L, McGinnis CS, McKinley KL, Rathnayake A, Vaka D, Nguyen V, Trentesaux C, Reyes E, Gupta AR, Gartner ZJ, Locksley RM, Gardner JM, Itzkovitz S, Boffelli D, Klein OD. 2023. Epithelial zonation along the mouse and human small intestine defines five discrete metabolic domains. bioRxiv : the preprint server for biology. Pubmed: 37790430 DOI:10.1101/2023.09.20.558726 A key aspect of nutrient absorption is the exquisite division of labor across the length of the small intestine, with individual classes of micronutrients taken up at different positions. For millennia, the small intestine was thought to comprise three segments with indefinite borders: the duodenum, jejunum, and ileum. By examining fine-scale longitudinal segmentation of the mouse and human small intestines, we identified transcriptional signatures and upstream regulatory factors that define five domains of nutrient absorption, distinct from the three traditional sections. Spatially restricted expression programs were most prominent in nutrient-absorbing enterocytes but initially arose in intestinal stem cells residing in three regional populations. While a core signature was maintained across mice and humans with different diets and environments, domain properties were influenced by dietary changes. We established the functions of and in patterning lipid metabolism in distal domains and generated a predictive model of additional transcription factors that direct domain identity. Molecular domain identity can be detected with machine learning, representing the first systematic method to computationally identify specific intestinal regions in mice. These findings provide a foundational framework for the identity and control of longitudinal zonation of absorption along the proximal:distal small intestinal axis. -
McKinley KL, Longaker MT, Naik S. 2023. Emerging frontiers in regenerative medicine. Science (New York, N.Y.). 380(6647):796-798. Pubmed: 37228215 DOI:10.1126/science.add6492 McKinley KL, Longaker MT, Naik S. 2023. Emerging frontiers in regenerative medicine. Science (New York, N.Y.). 380(6647):796-798. Pubmed: 37228215 DOI:10.1126/science.add6492 Bridging knowledge gaps could enable regenerative therapy. -
Bayin NS, McKinley KL, LaFave LM. 2023. Research vision workshopping: Peer mentoring to support the transition to independence. Cell. 186(7):1295-1299. Pubmed: 37001493 DOI:S0092-8674(23)00220-9 Bayin NS, McKinley KL, LaFave LM. 2023. Research vision workshopping: Peer mentoring to support the transition to independence. Cell. 186(7):1295-1299. Pubmed: 37001493 DOI:S0092-8674(23)00220-9 The transition to independence requires shared enthusiasm for one's research goals from broad audiences. In this commentary, we describe the use of "research vision workshopping" within peer mentoring networks. We contend that this approach is broadly useful for the development and refinement of research visions for the academic job search.Copyright © 2023 Elsevier Inc. All rights reserved. 2022
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McKinley KL, Didychuk AL, Nicholas DA, Termini CM. 2022. The transition phase: preparing to launch a laboratory. Trends in biochemical sciences. 47(10):814-818. Pubmed: 35644775 DOI:S0968-0004(22)00116-5 McKinley KL, Didychuk AL, Nicholas DA, Termini CM. 2022. The transition phase: preparing to launch a laboratory. Trends in biochemical sciences. 47(10):814-818. Pubmed: 35644775 DOI:S0968-0004(22)00116-5 The process of starting a laboratory varies between institutions. However, there are universal tasks all investigators will need to address when launching their laboratories. In this piece, we provide a brief summary of considerations for incoming group leaders to centralize this information for the scientific community.Copyright © 2022 Elsevier Ltd. All rights reserved. -
McKinley KL, van Boxtel R, Finley LWS, Peña CJ, Nowakowski TJ. 2022. New York Stem Cell Foundation Robertson Investigators. Cell stem cell. 29(12):1621-1623. Pubmed: 36459965 DOI:S1934-5909(22)00452-0 McKinley KL, van Boxtel R, Finley LWS, Peña CJ, Nowakowski TJ. 2022. New York Stem Cell Foundation Robertson Investigators. Cell stem cell. 29(12):1621-1623. Pubmed: 36459965 DOI:S1934-5909(22)00452-0 As the stem cell community mourns the loss of New York Stem Cell Foundation founder Susan Solomon, we also look to celebrate her legacy. In this Voices, members of the 2022 class of NYSCF Roberston Investigators share how NYSCF community support will impact them and the bold ideas they will pursue as a result.Copyright © 2022 Elsevier Inc. All rights reserved. 2020
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Smith Q, McKinley KL, Chan CJ, Sikandar S, Xiang Y, Passaro D. 2020. Introductions to the Community: Early-Career Researchers in the Time of COVID-19. Cell stem cell. 27(5):702-704. Pubmed: 33157046 DOI:S1934-5909(20)30502-6 Smith Q, McKinley KL, Chan CJ, Sikandar S, Xiang Y, Passaro D. 2020. Introductions to the Community: Early-Career Researchers in the Time of COVID-19. Cell stem cell. 27(5):702-704. Pubmed: 33157046 DOI:S1934-5909(20)30502-6 COVID-19 has unfortunately halted lab work, conferences, and in-person networking, which is especially detrimental to researchers just starting their labs. Through social media and our reviewer networks, we met some early-career stem cell investigators impacted by the closures. Here, they introduce themselves and their research to our readers.Copyright © 2020 Elsevier Inc. All rights reserved. -
Skokan TD, Vale RD, McKinley KL. 2020. Cell Sorting in Hydra vulgaris Arises from Differing Capacities for Epithelialization between Cell Types. Current biology : CB. 30(19):3713-3723.e3. Pubmed: 32795440 DOI:S0960-9822(20)31027-7 Skokan TD, Vale RD, McKinley KL. 2020. Cell Sorting in Hydra vulgaris Arises from Differing Capacities for Epithelialization between Cell Types. Current biology : CB. 30(19):3713-3723.e3. Pubmed: 32795440 DOI:S0960-9822(20)31027-7 Hydra vulgaris exhibits a remarkable capacity to reassemble its body plan from a disordered aggregate of cells. Reassembly begins by sorting two epithelial cell types, endoderm and ectoderm, into inner and outer layers, respectively. The cellular features and behaviors that distinguish ectodermal and endodermal lineages to drive sorting have not been fully elucidated. To dissect this process, we use micromanipulation to position single cells of diverse lineages on the surface of defined multicellular aggregates and monitor sorting outcomes by live imaging. Although sorting has previously been attributed to intrinsic differences between the epithelial lineages, we find that single cells of all lineages sort to the interior of ectodermal aggregates, including single ectodermal cells. This reveals that cells of the same lineage can adopt opposing positions when sorting as individuals or a collective. Ectodermal cell collectives adopt their position at the aggregate exterior by rapidly reforming an epithelium that engulfs cells adhered to its surface through a collective spreading behavior. In contrast, aggregated endodermal cells persistently lose epithelial features. These non-epithelialized aggregates, like isolated cells of all lineages, are adherent passengers for engulfment by the ectodermal epithelium. We find that collective spreading of the ectoderm and persistent de-epithelialization in the endoderm also arise during local wounding in Hydra, suggesting that Hydra's wound-healing and self-organization capabilities may employ similar mechanisms. Together, our data suggest that differing propensities for epithelialization can sort cell types into distinct compartments to build and restore complex tissue architecture.Copyright © 2020 Elsevier Inc. All rights reserved. -
McKinley KL, Castillo-Azofeifa D, Klein OD. 2020. Tools and Concepts for Interrogating and Defining Cellular Identity. Cell stem cell. 26(5):632-656. Pubmed: 32386555 DOI:S1934-5909(20)30137-5 McKinley KL, Castillo-Azofeifa D, Klein OD. 2020. Tools and Concepts for Interrogating and Defining Cellular Identity. Cell stem cell. 26(5):632-656. Pubmed: 32386555 DOI:S1934-5909(20)30137-5 Defining the mechanisms that generate specialized cell types and coordinate their functions is critical for understanding organ development and renewal. New tools and discoveries are challenging and refining our definitions of a cell type. A rapidly growing toolkit for single-cell analyses has expanded the number of markers that can be assigned to a cell simultaneously, revealing heterogeneity within cell types that were previously regarded as homogeneous populations. Additionally, cell types defined by specific molecular markers can exhibit distinct, context-dependent functions; for example, between tissues in homeostasis and those responding to damage. Here we review the current technologies used to identify and characterize cells, and we discuss how experimental and pathological perturbations are adding increasing complexity to our definitions of cell identity.Copyright © 2020 Elsevier Inc. All rights reserved. 2018
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Rodriguez-Rodriguez JA, Lewis C, McKinley KL, Sikirzhytski V, Corona J, Maciejowski J, Khodjakov A, Cheeseman IM, Jallepalli PV. 2018. Distinct Roles of RZZ and Bub1-KNL1 in Mitotic Checkpoint Signaling and Kinetochore Expansion. Current biology : CB. 28(21):3422-3429.e5. Pubmed: 30415700 DOI:S0960-9822(18)31335-6 Rodriguez-Rodriguez JA, Lewis C, McKinley KL, Sikirzhytski V, Corona J, Maciejowski J, Khodjakov A, Cheeseman IM, Jallepalli PV. 2018. Distinct Roles of RZZ and Bub1-KNL1 in Mitotic Checkpoint Signaling and Kinetochore Expansion. Current biology : CB. 28(21):3422-3429.e5. Pubmed: 30415700 DOI:S0960-9822(18)31335-6 The Mad1-Mad2 heterodimer is the catalytic hub of the spindle assembly checkpoint (SAC), which controls M phase progression through a multi-subunit anaphase inhibitor, the mitotic checkpoint complex (MCC) [1, 2]. During interphase, Mad1-Mad2 generates MCC at nuclear pores [3]. After nuclear envelope breakdown (NEBD), kinetochore-associated Mad1-Mad2 catalyzes MCC assembly until all chromosomes achieve bipolar attachment [1, 2]. Mad1-Mad2 and other factors are also incorporated into the fibrous corona, a phospho-dependent expansion of the outer kinetochore that precedes microtubule attachment [4-6]. The factor(s) involved in targeting Mad1-Mad2 to kinetochores in higher eukaryotes remain controversial [7-12], and the specific phosphorylation event(s) that trigger corona formation remain elusive [5, 13]. We used genome editing to eliminate Bub1, KNL1, and the Rod-Zw10-Zwilch (RZZ) complex in human cells. We show that RZZ's sole role in SAC activation is to tether Mad1-Mad2 to kinetochores. Separately, Mps1 kinase triggers fibrous corona formation by phosphorylating two N-terminal sites on Rod. In contrast, Bub1 and KNL1 activate kinetochore-bound Mad1-Mad2 to produce a "wait anaphase" signal but are not required for corona formation. We also show that clonal lines isolated after BUB1 disruption recover Bub1 expression and SAC function through nonsense-associated alternative splicing (NAS). Our study reveals a fundamental division of labor in the mammalian SAC and highlights a transcriptional response to nonsense mutations that can reduce or eliminate penetrance in genome editing experiments.Copyright © 2018 Elsevier Ltd. All rights reserved. -
McKinley KL. 2018. Employing CRISPR/Cas9 genome engineering to dissect the molecular requirements for mitosis. Methods in cell biology. 144:75-105. Pubmed: 29804684 DOI:S0091-679X(18)30003-7 McKinley KL. 2018. Employing CRISPR/Cas9 genome engineering to dissect the molecular requirements for mitosis. Methods in cell biology. 144:75-105. Pubmed: 29804684 DOI:S0091-679X(18)30003-7 The faithful execution of cell division requires the coordinated action of hundreds of gene products. Precisely perturbing these gene products in cells is central to understanding their functions during normal cell division, and the contributions of their disruption to disease. Here, we describe experimental approaches for using CRISPR/Cas9 for gene disruption and modification, with a focus on human cell culture. We describe strategies for inducible gene disruption to generate acute knockouts of essential cell division genes, which can be modified for the chronic elimination of nonessential genes. We also describe strategies for modifying the genome to generate protein fusions to report on and modify protein behavior. These tools facilitate investigation of protein function, dissection of protein assembly networks, and analyses of disease-associated mutations.© 2018 Elsevier Inc. All rights reserved. -
McKinley KL, Stuurman N, Royer LA, Schartner C, Castillo-Azofeifa D, Delling M, Klein OD, Vale RD. 2018. Cellular aspect ratio and cell division mechanics underlie the patterning of cell progeny in diverse mammalian epithelia. eLife. 7. Pubmed: 29897330 DOI:10.7554/eLife.36739 McKinley KL, Stuurman N, Royer LA, Schartner C, Castillo-Azofeifa D, Delling M, Klein OD, Vale RD. 2018. Cellular aspect ratio and cell division mechanics underlie the patterning of cell progeny in diverse mammalian epithelia. eLife. 7. Pubmed: 29897330 DOI:10.7554/eLife.36739 Cell division is essential to expand, shape, and replenish epithelia. In the adult small intestine, cells from a common progenitor intermix with other lineages, whereas cell progeny in many other epithelia form contiguous patches. The mechanisms that generate these distinct patterns of progeny are poorly understood. Using light sheet and confocal imaging of intestinal organoids, we show that lineages intersperse during cytokinesis, when elongated interphase cells insert between apically displaced daughters. Reducing the cellular aspect ratio to minimize the height difference between interphase and mitotic cells disrupts interspersion, producing contiguous patches. Cellular aspect ratio is similarly a key parameter for division-coupled interspersion in the early mouse embryo, suggesting that this physical mechanism for patterning progeny may pertain to many mammalian epithelia. Our results reveal that the process of cytokinesis in elongated mammalian epithelia allows lineages to intermix and that cellular aspect ratio is a critical modulator of the progeny pattern.© 2018, McKinley et al. 2017
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McKinley KL, Cheeseman IM. 2017. Large-Scale Analysis of CRISPR/Cas9 Cell-Cycle Knockouts Reveals the Diversity of p53-Dependent Responses to Cell-Cycle Defects. Developmental cell. 40(4):405-420.e2. Pubmed: 28216383 DOI:S1534-5807(17)30039-4 McKinley KL, Cheeseman IM. 2017. Large-Scale Analysis of CRISPR/Cas9 Cell-Cycle Knockouts Reveals the Diversity of p53-Dependent Responses to Cell-Cycle Defects. Developmental cell. 40(4):405-420.e2. Pubmed: 28216383 DOI:S1534-5807(17)30039-4 Defining the genes that are essential for cellular proliferation is critical for understanding organismal development and identifying high-value targets for disease therapies. However, the requirements for cell-cycle progression in human cells remain incompletely understood. To elucidate the consequences of acute and chronic elimination of cell-cycle proteins, we generated and characterized inducible CRISPR/Cas9 knockout human cell lines targeting 209 genes involved in diverse cell-cycle processes. We performed single-cell microscopic analyses to systematically establish the effects of the knockouts on subcellular architecture. To define variations in cell-cycle requirements between cultured cell lines, we generated knockouts across cell lines of diverse origins. We demonstrate that p53 modulates the phenotype of specific cell-cycle defects through distinct mechanisms, depending on the defect. This work provides a resource to broadly facilitate robust and long-term depletion of cell-cycle proteins and reveals insights into the requirements for cell-cycle progression.Copyright © 2017 Elsevier Inc. All rights reserved. -
Guo LY, Allu PK, Zandarashvili L, McKinley KL, Sekulic N, Dawicki-McKenna JM, Fachinetti D, Logsdon GA, Jamiolkowski RM, Cleveland DW, Cheeseman IM, Black BE. 2017. Centromeres are maintained by fastening CENP-A to DNA and directing an arginine anchor-dependent nucleosome transition. Nature communications. 8:15775. Pubmed: 28598437 DOI:10.1038/ncomms15775 Guo LY, Allu PK, Zandarashvili L, McKinley KL, Sekulic N, Dawicki-McKenna JM, Fachinetti D, Logsdon GA, Jamiolkowski RM, Cleveland DW, Cheeseman IM, Black BE. 2017. Centromeres are maintained by fastening CENP-A to DNA and directing an arginine anchor-dependent nucleosome transition. Nature communications. 8:15775. Pubmed: 28598437 DOI:10.1038/ncomms15775 Maintaining centromere identity relies upon the persistence of the epigenetic mark provided by the histone H3 variant, centromere protein A (CENP-A), but the molecular mechanisms that underlie its remarkable stability remain unclear. Here, we define the contributions of each of the three candidate CENP-A nucleosome-binding domains (two on CENP-C and one on CENP-N) to CENP-A stability using gene replacement and rapid protein degradation. Surprisingly, the most conserved domain, the CENP-C motif, is dispensable. Instead, the stability is conferred by the unfolded central domain of CENP-C and the folded N-terminal domain of CENP-N that becomes rigidified 1,000-fold upon crossbridging CENP-A and its adjacent nucleosomal DNA. Disrupting the 'arginine anchor' on CENP-C for the nucleosomal acidic patch disrupts the CENP-A nucleosome structural transition and removes CENP-A nucleosomes from centromeres. CENP-A nucleosome retention at centromeres requires a core centromeric nucleosome complex where CENP-C clamps down a stable nucleosome conformation and CENP-N fastens CENP-A to the DNA. 2016
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McKinley KL, Cheeseman IM. 2016. The molecular basis for centromere identity and function. Nature reviews. Molecular cell biology. 17(1):16-29. Pubmed: 26601620 DOI:10.1038/nrm.2015.5 McKinley KL, Cheeseman IM. 2016. The molecular basis for centromere identity and function. Nature reviews. Molecular cell biology. 17(1):16-29. Pubmed: 26601620 DOI:10.1038/nrm.2015.5 The centromere is the region of the chromosome that directs its segregation in mitosis and meiosis. Although the functional importance of the centromere has been appreciated for more than 130 years, elucidating the molecular features and properties that enable centromeres to orchestrate chromosome segregation is an ongoing challenge. Most eukaryotic centromeres are defined epigenetically and require the presence of nucleosomes containing the histone H3 variant centromere protein A (CENP-A; also known as CENH3). Ongoing work is providing important molecular insights into the central requirements for centromere identity and propagation, and the mechanisms by which centromeres recruit kinetochores to connect to spindle microtubules. 2015
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McKinley KL, Sekulic N, Guo LY, Tsinman T, Black BE, Cheeseman IM. 2015. The CENP-L-N Complex Forms a Critical Node in an Integrated Meshwork of Interactions at the Centromere-Kinetochore Interface. Molecular cell. 60(6):886-98. Pubmed: 26698661 DOI:S1097-2765(15)00818-7 McKinley KL, Sekulic N, Guo LY, Tsinman T, Black BE, Cheeseman IM. 2015. The CENP-L-N Complex Forms a Critical Node in an Integrated Meshwork of Interactions at the Centromere-Kinetochore Interface. Molecular cell. 60(6):886-98. Pubmed: 26698661 DOI:S1097-2765(15)00818-7 During mitosis, the macromolecular kinetochore complex assembles on the centromere to orchestrate chromosome segregation. The properties and architecture of the 16-subunit Constitutive Centromere-Associated Network (CCAN) that allow it to build a robust platform for kinetochore assembly are poorly understood. Here, we use inducible CRISPR knockouts and biochemical reconstitutions to define the interactions between the human CCAN proteins. We find that the CCAN does not assemble as a linear hierarchy, and instead, each sub-complex requires multiple non-redundant interactions for its localization to centromeres and the structural integrity of the overall assembly. We demonstrate that the CENP-L-N complex plays a crucial role at the core of this assembly through interactions with CENP-C and CENP-H-I-K-M. Finally, we show that the CCAN is remodeled over the cell cycle such that sub-complexes depend on their interactions differentially. Thus, an interdependent meshwork within the CCAN underlies the centromere specificity and stability of the kinetochore.Copyright © 2015 Elsevier Inc. All rights reserved. 2014
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McKinley KL, Cheeseman IM. 2014. Polo-like kinase 1 licenses CENP-A deposition at centromeres. Cell. 158(2):397-411. Pubmed: 25036634 DOI:S0092-8674(14)00748-X McKinley KL, Cheeseman IM. 2014. Polo-like kinase 1 licenses CENP-A deposition at centromeres. Cell. 158(2):397-411. Pubmed: 25036634 DOI:S0092-8674(14)00748-X To ensure the stable transmission of the genome during vertebrate cell division, the mitotic spindle must attach to a single locus on each chromosome, termed the centromere. The fundamental requirement for faithful centromere inheritance is the controlled deposition of the centromere-specifying histone, CENP-A. However, the regulatory mechanisms that ensure the precise control of CENP-A deposition have proven elusive. Here, we identify polo-like kinase 1 (Plk1) as a centromere-localized regulator required to initiate CENP-A deposition in human cells. We demonstrate that faithful CENP-A deposition requires integrated signals from Plk1 and cyclin-dependent kinase (CDK), with Plk1 promoting the localization of the key CENP-A deposition factor, the Mis18 complex, and CDK inhibiting Mis18 complex assembly. By bypassing these regulated steps, we uncoupled CENP-A deposition from cell-cycle progression, resulting in mitotic defects. Thus, CENP-A deposition is controlled by a two-step regulatory paradigm comprised of Plk1 and CDK that is crucial for genomic integrity.Copyright © 2014 Elsevier Inc. All rights reserved. -
Thiru P, Kern DM, McKinley KL, Monda JK, Rago F, Su KC, Tsinman T, Yarar D, Bell GW, Cheeseman IM. 2014. Kinetochore genes are coordinately up-regulated in human tumors as part of a FoxM1-related cell division program. Molecular biology of the cell. 25(13):1983-94. Pubmed: 24829384 DOI:10.1091/mbc.E14-03-0837 Thiru P, Kern DM, McKinley KL, Monda JK, Rago F, Su KC, Tsinman T, Yarar D, Bell GW, Cheeseman IM. 2014. Kinetochore genes are coordinately up-regulated in human tumors as part of a FoxM1-related cell division program. Molecular biology of the cell. 25(13):1983-94. Pubmed: 24829384 DOI:10.1091/mbc.E14-03-0837 The key player in directing proper chromosome segregation is the macromolecular kinetochore complex, which mediates DNA-microtubule interactions. Previous studies testing individual kinetochore genes documented examples of their overexpression in tumors relative to normal tissue, leading to proposals that up-regulation of specific kinetochore genes may promote tumor progression. However, kinetochore components do not function in isolation, and previous studies did not comprehensively compare the expression behavior of kinetochore components. Here we analyze the expression behavior of the full range of human kinetochore components in diverse published expression compendia, including normal tissues and tumor samples. Our results demonstrate that kinetochore genes are rarely overexpressed individually. Instead, we find that core kinetochore genes are coordinately regulated with other cell division genes under virtually all conditions. This expression pattern is strongly correlated with the expression of the forkhead transcription factor FoxM1, which binds to the majority of cell division promoters. These observations suggest that kinetochore gene up-regulation in cancer reflects a general activation of the cell division program and that altered expression of individual kinetochore genes is unlikely to play a causal role in tumorigenesis.© 2014 Thiru et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0). 2011
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Fisher MA, McKinley KL, Bradley LH, Viola SR, Hecht MH. 2011. De novo designed proteins from a library of artificial sequences function in Escherichia coli and enable cell growth. PloS one. 6(1):e15364. Pubmed: 21245923 DOI:10.1371/journal.pone.0015364 Fisher MA, McKinley KL, Bradley LH, Viola SR, Hecht MH. 2011. De novo designed proteins from a library of artificial sequences function in Escherichia coli and enable cell growth. PloS one. 6(1):e15364. Pubmed: 21245923 DOI:10.1371/journal.pone.0015364 A central challenge of synthetic biology is to enable the growth of living systems using parts that are not derived from nature, but designed and synthesized in the laboratory. As an initial step toward achieving this goal, we probed the ability of a collection of >10(6) de novo designed proteins to provide biological functions necessary to sustain cell growth. Our collection of proteins was drawn from a combinatorial library of 102-residue sequences, designed by binary patterning of polar and nonpolar residues to fold into stable 4-helix bundles. We probed the capacity of proteins from this library to function in vivo by testing their abilities to rescue 27 different knockout strains of Escherichia coli, each deleted for a conditionally essential gene. Four different strains--ΔserB, ΔgltA, ΔilvA, and Δfes--were rescued by specific sequences from our library. Further experiments demonstrated that a strain simultaneously deleted for all four genes was rescued by co-expression of four novel sequences. Thus, cells deleted for ∼0.1% of the E. coli genome (and ∼1% of the genes required for growth under nutrient-poor conditions) can be sustained by sequences designed de novo.