Bio

Honors & Awards


  • Leukemia and Lymphoma Society Postdoctoral Fellowship, Leukemia and Lymphoma Society (2014-present)
  • NSERC postdoctoral fellowship (declined), Natural Sciences and Engineering Research Council of Canada (2014)
  • Stanford University School of Medicine Dean’s Fellowship, Stanford School of Medicine (2014)
  • Faculty of Science Doctoral Dissertation Award, University of Alberta (2012)
  • Queen Elizabeth II Graduate Scholarship, University of Alberta (2011-2012)
  • PhD Graduate Student Scholarship, Alberta Innovates (2009-2011)
  • Michael Smith Foreign Study Supplement, Natural Sciences and Engineering Research Council of Canada (2009-2010)
  • President’s Prize of Distinction, University of Alberta (2009)
  • Alexander Graham Bell Canada Graduate Scholarship, Natural Sciences and Engineering Research Council of Canada (2008-2011)
  • President’s Prize of Distinction, University of Alberta (2008)
  • Provost Doctoral Entrance Scholarship, University of Alberta (2007)
  • Michael Smith Foundation Fellowship for Health Research, Michael Smith Foundation (2006-2007)
  • Andy Farquharson Award for Excellence in Graduate Student Teaching, University of Victoria (2006)
  • Dr. Julius F. Schleicher Graduate Scholarship, University of Victoria (2006)

Professional Education


  • Bachelor of Science, University of Victoria (2005)
  • Doctor of Philosophy, University of Alberta (2012)
  • Master of Science, University of Victoria (2007)

Stanford Advisors


Research & Scholarship

Lab Affiliations


Publications

All Publications


  • Ratiometric biosensors based on dimerization-dependent fluorescent protein exchange. Nature methods Ding, Y., Li, J., Enterina, J. R., Shen, Y., Zhang, I., Tewson, P. H., Mo, G. C., Zhang, J., Quinn, A. M., Hughes, T. E., Maysinger, D., Alford, S. C., Zhang, Y., Campbell, R. E. 2015; 12 (3): 195-198

    Abstract

    We have developed a versatile new class of genetically encoded fluorescent biosensor based on reversible exchange of the heterodimeric partners of green and red dimerization-dependent fluorescent proteins. We demonstrate the use of this strategy to construct both intermolecular and intramolecular ratiometric biosensors for qualitative imaging of caspase activity, Ca(2+) concentration dynamics and other second-messenger signaling activities.

    View details for DOI 10.1038/nmeth.3261

    View details for PubMedID 25622108

  • Conditional protein splicing of alpha-sarcin in live cells MOLECULAR BIOSYSTEMS Alford, S. C., O'Sullivan, C., Obst, J., Christie, J., Howard, P. L. 2014; 10 (4): 831-837

    Abstract

    Protein splicing technology harnesses the ability of inteins to ligate protein fragments, forming a mature protein. This report describes our effort to engineer rapamycin-dependent protein splicing of a ribotoxin, called α-sarcin. Engineering this system required the investigation of important splicing parameters, including extein context and splicing temperature. We show α-sarcin splicing is dependent on rapamycin, is inducible with rapid kinetics, and triggers apoptosis in HeLa cells. These findings establish a proof-of-concept for a conditional cell ablation strategy.

    View details for DOI 10.1039/c3mb70387h

    View details for Web of Science ID 000332456200014

    View details for PubMedID 24481070

  • Optogenetic reporters BIOLOGY OF THE CELL Alford, S. C., Wu, J., Zhao, Y., Campbell, R. E., Knoepfel, T. 2013; 105 (1): 14-29

    Abstract

    The discovery of naturally evolved fluorescent proteins and their subsequent tuning by protein engineering provided the basis for a large family of genetically encoded biosensors that report a variety of physicochemical processes occurring in living tissue. These optogenetic reporters are powerful tools for live-cell microscopy and quantitative analysis at the subcellular level. In this review, we present an overview of the transduction mechanisms that have been exploited for engineering these genetically encoded reporters. Finally, we discuss current and future efforts towards the combined use of various optogenetic actuators and reporters for simultaneously controlling and imaging the physiology of cells and tissues.

    View details for DOI 10.1111/boc.201200054

    View details for Web of Science ID 000312990400004

    View details for PubMedID 23126299

  • Dimerization-Dependent Green and Yellow Fluorescent Proteins ACS SYNTHETIC BIOLOGY Alford, S. C., Ding, Y., Simmen, T., Campbell, R. E. 2012; 1 (12): 569-575

    Abstract

    Dimerization-dependent fluorescent proteins (ddFP) are a recently introduced class of genetically encoded reporters that can be used for the detection of protein interactions in live cells. The progenitor of this class of tools was a red fluorescent ddFP (ddRFP) derived from a homodimeric variant of Discosoma red fluorescent protein. Here, we describe the engineering and application of an expanded palette of ddFPs, which includes green (ddGFP) and yellow (ddYFP) variants. These optimized variants offer several advantages relative to ddRFP including increased in vitro contrast and brightness for ddGFP and increased brightness and a lowered pK a for ddYFP. We demonstrate that both variants are useful as biosensors for protease activity in live cells. Using the ddGFP tool, we generated a highly effective indicator of endomembrane proximity that can be used to image the mitochondria-associated membrane (MAM) interface of endoplasmic reticulum (ER) and mitochondria.

    View details for DOI 10.1021/sb300050j

    View details for Web of Science ID 000312679800001

    View details for PubMedID 23656278

  • A Fluorogenic Red Fluorescent Protein Heterodimer CHEMISTRY & BIOLOGY Alford, S. C., Abdelfattah, A. S., Ding, Y., Campbell, R. E. 2012; 19 (3): 353-360

    Abstract

    The expanding repertoire of genetically encoded biosensors constructed from variants of Aequorea victoria green fluorescent protein (GFP) enable the imaging of a variety of intracellular biochemical processes. To facilitate the imaging of multiple biosensors in a single cell, we undertook the development of a dimerization-dependent red fluorescent protein (ddRFP) that provides an alternative strategy for biosensor construction. An extensive process of rational engineering and directed protein evolution led to the discovery of a ddRFP with a K(d) of 33 μM and a 10-fold increase in fluorescence upon heterodimer formation. We demonstrate that the dimerization-dependent fluorescence of ddRFP can be used for detection of a protein-protein interaction in vitro, imaging of the reversible Ca²⁺-dependent association of calmodulin and M13 in live cells, and imaging of caspase-3 activity during apoptosis.

    View details for DOI 10.1016/j.chembiol.2012.01.006

    View details for Web of Science ID 000302588900008

    View details for PubMedID 22444590

  • Soluble ephrin a1 is necessary for the growth of HeLa and SK-BR3 cells CANCER CELL INTERNATIONAL Alford, S., Watson-Hurthig, A., Scott, N., Carette, A., Lorimer, H., Bazowski, J., Howard, P. L. 2010; 10

    Abstract

    Ephrin A1 (EFNA1) is a member of the A-type ephrin family of cell surface proteins that function as ligands for the A-type Eph receptor tyrosine kinase family. In malignancy, the precise role of EFNA1 and its preferred receptor, EPHA2, is controversial. Several studies have found that EFNA1 may suppress EPHA2-mediated oncogenesis, or enhance it, depending on cell type and context. However, little is known about the conditions that influence whether EFNA1 promotes or suppresses tumorigenicity. EFNA1 exists in a soluble form as well as a glycophosphatidylinositol (GPI) membrane attached form. We investigated whether the contradictory roles of EFNA1 in malignancy might in part be related to the existence of both soluble and membrane attached forms of EFNA1 and potential differences in the manner in which they interact with EPHA2.Using a RNAi strategy to reduce the expression of endogenous EFNA1 and EPHA2, we found that both EFNA1 and EPHA2 are required for growth of HeLa and SK-BR3 cells. The growth defects could be rescued by conditioned media from cells overexpressing soluble EFNA1. Interestingly, we found that overexpression of the membrane attached form of EFNA1 suppresses growth of HeLa cells in 3D but not 2D. Knockdown of endogenous EFNA1, or overexpression of full-length EFNA1, resulted in relocalization of EPHA2 from the cell surface to sites of cell-cell contact. Overexpression of soluble EFNA1 however resulted in more EPHA2 distributed on the cell surface, away from cell-cell contacts, and promoted the growth of HeLa cells.We conclude that soluble EFNA1 is necessary for the transformation of HeLa and SK-BR3 cells and participates in the relocalization of EPHA2 away from sites of cell-cell contact during transformation.

    View details for DOI 10.1186/1475-2867-10-41

    View details for Web of Science ID 000284089200001

    View details for PubMedID 20979646

  • alpha-Sarcin catalytic activity is not required for cytotoxicity BMC BIOCHEMISTRY Alford, S. C., Pearson, J. D., Carette, A., Ingham, R. J., Howard, P. L. 2009; 10

    Abstract

    Alpha-sarcin is a protein toxin produced by Aspergillus giganteus. It belongs to a family of cytotoxic ribonucleases that inactivate the ribosome and inhibit protein synthesis. alpha-Sarcin cleaves a single phosphodiester bond within the RNA backbone of the large ribosomal subunit, which makes the ribosome unrecognizable to elongation factors and, in turn, blocks protein synthesis. Although it is widely held that the protein synthesis inhibition caused by the toxin leads to cell death, it has not been directly shown that catalytically inactive mutants of alpha-sarcin are non-toxic when expressed directly within the cytoplasm of cells. This is important since recent studies have cast doubt on whether protein synthesis inhibition is sufficient to initiate apoptosis.In this report, we assay alpha-sarcin cytotoxicity and ability to inhibit protein synthesis by direct cytoplasmic expression. We show that mutations in alpha-sarcin, which impair alpha-sarcin's ability to inhibit protein synthesis, do not affect its cytotoxicity. The mutants are unable to activate JNK, confirming that the sarcin-ricin loop remains intact and that the alpha-sarcin mutants are catalytically inactive. In addition, both mutant and wildtype variants of alpha-sarcin localize to the nucleus and cytoplasm, where they co-localize with ribosomal marker RPS6.We conclude that although protein synthesis inhibition likely contributes to cell death, it is not required. Thus, our results suggest that alpha-sarcin can promote cell death through a previously unappreciated mechanism that is independent of rRNA cleavage and JNK activation.

    View details for DOI 10.1186/1471-2091-10-9

    View details for Web of Science ID 000265601500001

    View details for PubMedID 19344516

  • Tissue transglutaminase clusters soluble A-type ephrins into functionally active high molecular weight oligomers EXPERIMENTAL CELL RESEARCH Alford, S. C., Bazowski, J., Lorimer, H., Elowe, S., Howard, P. L. 2007; 313 (20): 4170-4179

    Abstract

    The Eph receptors and their ligands, the ephrins, are thought to act at points of close cell-cell contact to elicit bi-directional signaling in receptor and ligand expressing cells. However, when cultured in vitro, some A-type ephrins are released from the cell surface and it is unclear if these soluble ephrins participate in Eph receptor activation. We show that soluble ephrin A5 is subject to oligomerization. Ephrins A1 and A5 are substrates for a cross-linking enzyme, tissue transglutaminase, which mediates the formation of oligomeric ephrin. Transglutaminase-cross-linked ephrin binds to A-type Eph receptors, stimulates Eph kinase activity, and promotes invasion and migration of HeLa cells. Transglutaminase-mediated oligomerization of soluble ephrin potentially represents a novel mechanism of forward signaling through Eph receptors and may extend the influence of A-type ephrins beyond cell contact mediated signaling.

    View details for DOI 10.1016/j.yexcr.2007.07.019

    View details for Web of Science ID 000251358200008

    View details for PubMedID 17707797

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