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Superresolution Imaging Identifies That Conventional Trafficking Pathways Are Not Essential For Endoplasmic Reticulum To Outer Mitochondrial Membrane Protein Transport., Kyle Salka, Shivaprasad Bhuvanendran, Kassandra Wilson, Petros Bozidis, Mansi Mehta, Kristin Rainey, Hiromi Sesaki, George H Patterson, Jyoti K. Jaiswal, Anamaris M. Colberg-Poley 2017 George Washington University

Superresolution Imaging Identifies That Conventional Trafficking Pathways Are Not Essential For Endoplasmic Reticulum To Outer Mitochondrial Membrane Protein Transport., Kyle Salka, Shivaprasad Bhuvanendran, Kassandra Wilson, Petros Bozidis, Mansi Mehta, Kristin Rainey, Hiromi Sesaki, George H Patterson, Jyoti K. Jaiswal, Anamaris M. Colberg-Poley

Integrative Systems Biology Faculty Publications

Most nuclear-encoded mitochondrial proteins traffic from the cytosol to mitochondria. Some of these proteins localize at mitochondria-associated membranes (MAM), where mitochondria are closely apposed with the endoplasmic reticulum (ER). We have previously shown that the human cytomegalovirus signal-anchored protein known as viral mitochondria-localized inhibitor of apoptosis (vMIA) traffics from the ER to mitochondria and clusters at the outer mitochondrial membrane (OMM). Here, we have examined the host pathways by which vMIA traffics from the ER to mitochondria and clusters at the OMM. By disruption of phosphofurin acidic cluster sorting protein 2 (PACS-2), mitofusins (Mfn1/2), and dynamin related protein 1 ...


Stem Cell Derived Osteoprogenitors And Their Role In Bone Repair Using Morphogenic Activators, Angelina Miller, Sylvanna Islas, George Washington Brownridge III, Warren Hoeffler 2017 Dominican University of California

Stem Cell Derived Osteoprogenitors And Their Role In Bone Repair Using Morphogenic Activators, Angelina Miller, Sylvanna Islas, George Washington Brownridge Iii, Warren Hoeffler

Scholarly & Creative Works Conference 2017

Bone constantly cycles through a dynamic process of breakdown and remodeling. Osteoblasts are the specialized mesenchymal stem cells that have a major role in bone formation and the remodeling process whereas their counterpart osteoclasts, handle bone resorption. Embryonic stem cells can be partially differentiated into Progenitor cells, and we worked with #18, a candidate for being an osteoprogenitor that has the potential to respond to morphogenic activators. In the case of bone remodeling, TGF-β 2, BMP-2 and an abundance of CA++ have been shown to be potential activators of differentiation into osteoblasts. Eight different trials were conducted with the cells ...


A Sumo-Targeted Ubiquitin Ligase Is Involved In The Degradation Of The Nuclear Pool Of The Sumo E3 Ligase Siz1, Oliver Kerscher, Jason W. Westerbeck, Nagesh Pasupala, Mark Guillotte, Eva Szymanski, Brooke C. Matson, Cecilia Esteban 2017 College of William and Mary

A Sumo-Targeted Ubiquitin Ligase Is Involved In The Degradation Of The Nuclear Pool Of The Sumo E3 Ligase Siz1, Oliver Kerscher, Jason W. Westerbeck, Nagesh Pasupala, Mark Guillotte, Eva Szymanski, Brooke C. Matson, Cecilia Esteban

Oliver Kerscher

The Slx5/Slx8 heterodimer constitutes a SUMO-targeted ubiquitin ligase (STUbL) with an important role in SUMO-targeted degradation and SUMO-dependent signaling. This STUbL relies on SUMO-interacting motifs in Slx5 to aid in substrate targeting and carboxy-terminal RING domains in both Slx5 and Slx8 for substrate ubiquitylation. In budding yeast cells, Slx5 resides in the nucleus, forms distinct foci, and can associate with double-stranded DNA breaks. However, it remains unclear how STUbLs interact with other proteins and their substrates. To examine the targeting and functions of the Slx5/Slx8 STUbL, we constructed and analyzed truncations of the Slx5 protein. Our structure–function ...


Budding Yeast Protein Extraction And Purification For The Study Of Function, Interactions, And Post-Translational Modifications, Eva P. Szymanski, Oliver Kerscher 2017 College of William and Mary

Budding Yeast Protein Extraction And Purification For The Study Of Function, Interactions, And Post-Translational Modifications, Eva P. Szymanski, Oliver Kerscher

Oliver Kerscher

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome ...


Sumo-Targeted Ubiquitin Ligase (Stubl) Slx5 Regulates Proteolysis Of Centromeric Histone H3 Variant Cse4 And Prevents Its Mislocalization To Euchromatin, Kentaro Ohkuni, Yoshimitsu Takahashi, Alyona Fulp, Josh Lawrimore, Wei-Chun Au, Nagesh Pasupala, Reuben Levy-Myers, Jack Warren, Alexander Strunnikov, Richard E. Baker, Oliver Kerscher, Kerry Bloom, Munira A. Basrai 2017 National Cancer Institute

Sumo-Targeted Ubiquitin Ligase (Stubl) Slx5 Regulates Proteolysis Of Centromeric Histone H3 Variant Cse4 And Prevents Its Mislocalization To Euchromatin, Kentaro Ohkuni, Yoshimitsu Takahashi, Alyona Fulp, Josh Lawrimore, Wei-Chun Au, Nagesh Pasupala, Reuben Levy-Myers, Jack Warren, Alexander Strunnikov, Richard E. Baker, Oliver Kerscher, Kerry Bloom, Munira A. Basrai

Oliver Kerscher

Centromeric histone H3, CENP-ACse4, is essential for faithful chromosome segregation. Stringent regulation of cellular levels of CENP-ACse4 restricts its localization to centromeres. Mislocalization of CENP-ACse4 is associated with aneuploidy in yeast, flies and tumorigenesis in human cells; thus, defining pathways that regulate CENP-A levels is critical for understanding how mislocalization of CENP-A contributes to aneuploidy in human cancers. Previous work in budding yeast has shown that ubiquitination of overexpressed Cse4 by Psh1, an E3 ligase, partially contributes to proteolysis of Cse4. Here, we provide the first evidence that Cse4 is sumoylated by E3 ligases Siz1 and Siz2 in vivo and ...


Tim18p Is A New Component Of The Tim54p-Tim22p Translocon In The Mitochondrial Inner Membrane, Oliver Kerscher, Naresh B. Sepuri, Robert E. Jensen 2017 College of William and Mary

Tim18p Is A New Component Of The Tim54p-Tim22p Translocon In The Mitochondrial Inner Membrane, Oliver Kerscher, Naresh B. Sepuri, Robert E. Jensen

Oliver Kerscher

The mitochondrial inner membrane contains two separate translocons: one required for the translocation of matrix-targeted proteins (the Tim23p-Tim17p complex) and one for the insertion of polytopic proteins into the mitochondrial inner membrane (the Tim54p-Tim22p complex). To identify new members of the Tim54p-Tim22p complex, we screened for high-copy suppressors of the temperature-sensitivetim54-1 mutant. We identified a new gene,TIM18, that encodes an integral protein of the inner membrane. The following genetic and biochemical observations suggest that the Tim18 protein is part of the Tim54p-Tim22p complex in the inner membrane: multiple copies of TIM18 suppress thetim54-1 growth defect; thetim18::HIS3 disruption is ...


Recognizing Chromosomes In Trouble: Association Of The Spindle Checkpoint Protein Bub3p With Altered Kinetochores And A Unique Defective Centromere, Oliver Kerscher, Luciana B. Crotti, Munira A. Basrai 2017 College of William and Mary

Recognizing Chromosomes In Trouble: Association Of The Spindle Checkpoint Protein Bub3p With Altered Kinetochores And A Unique Defective Centromere, Oliver Kerscher, Luciana B. Crotti, Munira A. Basrai

Oliver Kerscher

Spindle checkpoint proteins monitor the interaction of the spindle apparatus with the kinetochores, halting anaphase even if the microtubule attachment of only a single chromosome is altered. In this study, we show that Bub3p of Saccharomyces cerevisiae, an evolutionarily conserved spindle checkpoint protein, exhibits distinct interactions with an altered or defective kinetochore(s). We show for the first time that green fluorescent protein-tagged S. cerevisiae Bub3p (Bub3-GFP) exhibits not only a diffuse nuclear localization pattern but also forms distinct nuclear foci in unperturbed growing and G2/M-arrested cells. As Bub3-GFP foci overlap only a subset of kinetochores, we tested ...


Sumo-Targeted Ubiquitin Ligase (Stubl) Slx5 Regulates Proteolysis Of Centromeric Histone H3 Variant Cse4 And Prevents Its Mislocalization To Euchromatin, Kentaro Ohkuni, Yoshimitsu Takahashi, Alyona Flup, Josh La, Wei-Chun Au, Nagesh Pasupala, Ruben Levy-Meyers, Jack Warren, Alexander Strunnikov, Richard E. Baker, Oliver Kerscher, Kerry Bloom, Munira A. Basrai 2017 College of William and Mary

Sumo-Targeted Ubiquitin Ligase (Stubl) Slx5 Regulates Proteolysis Of Centromeric Histone H3 Variant Cse4 And Prevents Its Mislocalization To Euchromatin, Kentaro Ohkuni, Yoshimitsu Takahashi, Alyona Flup, Josh La, Wei-Chun Au, Nagesh Pasupala, Ruben Levy-Meyers, Jack Warren, Alexander Strunnikov, Richard E. Baker, Oliver Kerscher, Kerry Bloom, Munira A. Basrai

Oliver Kerscher

Centromeric histone H3, CENP-ACse4, is essential for faithful chromosome segregation. Stringent regulation of cellular levels of CENP-ACse4 restricts its localization to centromeres. Mislocalization of CENP-ACse4 is associated with aneuploidy in yeast and flies and tumorigenesis in human cells; thus defining pathways that regulate CENP-A levels is critical for understanding how mislocalization of CENP-A contributes to aneuploidy in human cancers. Previous work in budding yeast shows that ubiquitination of overexpressed Cse4 by Psh1, an E3 ligase, partially contributes to proteolysis of Cse4. Here we provide the first evidence that Cse4 is sumoylated by E3 ligases Siz1 and Siz2 in vivo and ...


The Yeast Hex3·Slx8 Heterodimer Is A Ubiquitin Ligase Stimulated By Substrate Sumoylation, Yang Xie, Oliver Kerscher, Mary B. Kroetz, Heather F. McConchie, Patrick Sung, Mark Hochstrasser 2017 College of William and Mary

The Yeast Hex3·Slx8 Heterodimer Is A Ubiquitin Ligase Stimulated By Substrate Sumoylation, Yang Xie, Oliver Kerscher, Mary B. Kroetz, Heather F. Mcconchie, Patrick Sung, Mark Hochstrasser

Oliver Kerscher

Hex3 and Slx8 are Saccharomyces cerevisiae proteins with important functions in DNA damage control and maintenance of genomic stability. Both proteins have RING domains at their C termini. Such domains are common in ubiquitin and ubiquitin-like protein ligases (E3s), but little was known about the molecular functions of either protein. In this study we identified HEX3 as a high-copy suppressor of a temperature-sensitive small ubiquitin-related modifier (SUMO) protease mutant, ulp1ts, suggesting that it may affect cellular SUMO dynamics. Remarkably, even a complete deletion of ULP1 is strongly suppressed. Hex3 forms a heterodimer with Slx8. We found that the Hex3·Slx8 ...


The Yeast Nuclear Pore Complex Functionally Interacts With Components Of The Spindle Assembly Checkpoint, Tatiana Louk, Oliver Kerscher, Robert J. Scott, Munira A. Basrai, Richard W. Wozniak 2017 College of William and Mary

The Yeast Nuclear Pore Complex Functionally Interacts With Components Of The Spindle Assembly Checkpoint, Tatiana Louk, Oliver Kerscher, Robert J. Scott, Munira A. Basrai, Richard W. Wozniak

Oliver Kerscher

Aphysical and functional link between the nuclear pore complex (NPC) and the spindle checkpoint machinery has been established in the yeast Saccharomyces cerevisiae. We show that two proteins required for the execution of the spindle checkpoint, Mad1p and Mad2p, reside predominantly at the NPC throughout the cell cycle. There they are associated with a subcomplex of nucleoporins containing Nup53p, Nup170p, and Nup157p. The association of the Mad1p–Mad2p complex with the NPC requires Mad1p and is mediated in part by Nup53p. On activation of the spindle checkpoint, we detect changes in the interactions between these proteins, including the release of ...


A Sumo-Targeted Ubiquitin Ligase Is Involved In The Degradation Of The Nuclear Pool Of The Sumo E3 Ligase Siz1, Jason W. Westerbeck, Nagesh Pasupala, Mark Guillotte, Eva Szymanski, Brooke C. Matson, Cecilia Esteban, Oliver Kerscher 2017 College of William and Mary

A Sumo-Targeted Ubiquitin Ligase Is Involved In The Degradation Of The Nuclear Pool Of The Sumo E3 Ligase Siz1, Jason W. Westerbeck, Nagesh Pasupala, Mark Guillotte, Eva Szymanski, Brooke C. Matson, Cecilia Esteban, Oliver Kerscher

Oliver Kerscher

The Slx5/Slx8 heterodimer constitutes a SUMO-targeted ubiquitin ligase (STUbL) with an important role in SUMO-targeted degradation and SUMO-dependent signaling. This STUbL relies on SUMO-interacting motifs in Slx5 to aid in substrate targeting and carboxy-terminal RING domains in both Slx5 and Slx8 for substrate ubiquitylation. In budding yeast cells, Slx5 resides in the nucleus, forms distinct foci, and can associate with double-stranded DNA breaks. However, it remains unclear how STUbLs interact with other proteins and their substrates. To examine the targeting and functions of the Slx5/Slx8 STUbL, we constructed and analyzed truncations of the Slx5 protein. Our structure–function ...


Do You Really Want A Hunter-Gatherer Microbiota? Perils And Pitfalls For Your Gut, Michael Ruscio 2017 Dr. Michael Ruscio

Do You Really Want A Hunter-Gatherer Microbiota? Perils And Pitfalls For Your Gut, Michael Ruscio

Journal of Evolution and Health

Clinicians have long understood the immense impact the health of our gut has on things like weight, energy, depression, autoimmunity and more. With the recent advancements in microbiota research we have learned much more about this connection. Like never before we understand how we can alleviate chronic health conditions and enhance well-being through improving the health of the gut.

However, with this increased research has also come over speculation and ill-informed recommendations. This has high impact on today's healthcare professional or consumer who could unintentionally do harm. For example by replicating the microbiota of hunter-gatherers of Africa, we could ...


Functional Redundancy Between Trans-Golgi Network Snare Family Members In Arabidopsis Thaliana, Sang-Jin Kim, Diane C. Bassham 2017 Iowa State University

Functional Redundancy Between Trans-Golgi Network Snare Family Members In Arabidopsis Thaliana, Sang-Jin Kim, Diane C. Bassham

Diane Bassham

Background

Vesicle fusion is an essential process for maintaining the structure and function of the endomembrane system. Fusion is mediated by t-SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) fusion proteins on the target membrane and v-SNAREs on the vesicle membrane; v-and t-SNAREs interact with each other, driving vesicle fusion with the target membrane. The Arabidopsis thaliana trans-Golgi network resident SNAREs SYP41 and VTI12, along with YKT61/62, have been shown to function in vesicle fusion in vitro, consistent with immunoprecipitation results showing their interaction in Arabidopsis cell extracts. Conflicting published results have indicated that SYP4 family members are ...


Guidelines For The Use And Interpretation Of Assays For Monitoring Autophagy In Higher Eukaryotes, Daniel J. Klionsky, Michael J. Lenardo, Diane C. Bassham 2017 University of Michigan-Ann Arbor

Guidelines For The Use And Interpretation Of Assays For Monitoring Autophagy In Higher Eukaryotes, Daniel J. Klionsky, Michael J. Lenardo, Diane C. Bassham

Diane Bassham

Research in autophagy continues to accelerate,1 and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.2,3 There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there ...


Autophagy In Plants And Algae, Diane C. Bassham, Jose L. Crespo 2017 Iowa State University

Autophagy In Plants And Algae, Diane C. Bassham, Jose L. Crespo

Diane Bassham

Autophagy is a major cellular degradation pathway in which materials are delivered to the vacuole in double-membrane vesicles known as autophagosomes, broken down, and recycled (Li and Vierstra, 2012; Liu and Bassham, 2012). In photosynthetic organisms, the pathway is strongly activated by biotic and abiotic stresses, including nutrient limitation, oxidative, salt and drought stress and pathogen infection, and during senescence (Perez-Perez et al., 2012; Lv et al., 2014). Mutation of genes required for autophagy causes hypersensitivity to stress, indicating that autophagy is important for tolerance of multiple stresses. While autophagy is often non-selective, a growing number of examples of selectivity ...


Guidelines For The Use And Interpretation Of Assays For Monitoring Autophagy, Daniel J. Klionsky, Shoba Amarnath, Vellareddy Anantharam, Diane C. Bassham, Bryan Bellaire 2017 University of Michigan-Ann Arbor

Guidelines For The Use And Interpretation Of Assays For Monitoring Autophagy, Daniel J. Klionsky, Shoba Amarnath, Vellareddy Anantharam, Diane C. Bassham, Bryan Bellaire

Diane Bassham

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is ...


Characterization Of A New Oda3 Allele, Oda3-6, Defective In Assembly Of The Outer Dynein Arm-Docking Complex In Chlamydomonas Reinhardtii, Jason M. Brown, Matthew Mosley, Daniela Montes-Berrueta, Yuqing Hou, Fan Yang, Chasity Scarbrough, George B. Witman, Maureen Wirschell 2017 Salem State University

Characterization Of A New Oda3 Allele, Oda3-6, Defective In Assembly Of The Outer Dynein Arm-Docking Complex In Chlamydomonas Reinhardtii, Jason M. Brown, Matthew Mosley, Daniela Montes-Berrueta, Yuqing Hou, Fan Yang, Chasity Scarbrough, George B. Witman, Maureen Wirschell

Biology Faculty Publications

We have used an insertional mutagenesis approach to generate new C. reinhardtii motility mutants. Of 56 mutants isolated, one is a new allele at the ODA3 locus, called oda3-6. Similar to the previously characterized oda3 alleles, oda3-6 has a slow-jerky swimming phenotype and reduced swimming speed. The oda3-6 mutant fails to assemble the outer dynein arm motor and outer dynein arm—docking complex (ODA-DC) in the ciliary axoneme due to an insertion in the 5’ end of the DCC1 gene, which encodes the DC1 subunit of the ODA-DC. Transformation of oda3-6 with the wild-type DCC1 gene rescues the mutant swimming ...


P32. Altered Sensory Processing In Response To Novel Dreadd-Induced Inactivation Of Gaba In Pedunculopontine Tegmental Nucleus, Niveen Fulcher 2017 Western University

P32. Altered Sensory Processing In Response To Novel Dreadd-Induced Inactivation Of Gaba In Pedunculopontine Tegmental Nucleus, Niveen Fulcher

Western Research Forum

Niveen Fulcher1, Cleusa De Oliveira2, & Susanne Schmid1,2

1Schulich School of Medicine & Dentistry, University of Western Ontario

2Anatomy and Cell Biology, University of Western Ontario

Altered sensory processing in response to novel DREADD-induced inactivation of GABA in pedunculopontine tegmental nucleus

Background: Sensory processing deficits are associated with certain psychiatric illnesses, such as schizophrenia and autism spectrum disorder (ASD). Sensory filtering and sensorimotor gating are evolutionarily conserved preattentive responses that filter and block redundant sensory stimuli that would otherwise overwhelm our brains. To date, underlying mechanisms of these deficits are undefined. Prepulse inhibition (PPI) of the ...


P23. Elucidation Of The Signaling Pathway Of Mertk, Kyle Taruc 2017 Western University

P23. Elucidation Of The Signaling Pathway Of Mertk, Kyle Taruc

Western Research Forum

Background

Mer Tyrosine Kinase (MERTK) is a receptor which mediates efferocytosis – removal of apoptotic cells by phagocytes such as macrophages. MERTK plays a critical role in homeostasis, with mutations in MERTK associated with the development and progression of atherosclerosis, the buildup of cholesterol-laden plaque in the sub-arterial space. Indeed, one MERTK allele is associated with 66% (heterozygous) to 75% (homozygous) protection from atherosclerosis, while other alleles and SNP’s pre-dispose to atherosclerosis and autoimmunity. Complications resulting from atherosclerosis, including heart attack and stroke, are currently the second leading cause of mortality in Canada. Despite the importance of MERTK in atherosclerosis ...


Pulmonary Surfactant Fortified With Cath-2 As A Novel Therapy For Bacterial Pneumonia, Brandon J. Baer 2017 University of Western Ontario

Pulmonary Surfactant Fortified With Cath-2 As A Novel Therapy For Bacterial Pneumonia, Brandon J. Baer

Western Research Forum

Background: Bacterial pneumonia is a leading cause of death worldwide, with high mortality rates persisting even after antibiotic treatment. Current treatments for pneumonia involve administration of antibiotics, however after the bacteria are killed they release toxic substances that induce inflammation and lung dysfunction. Host defense peptides represent a potential solution to this problem through their ability to down regulate inflammation. However, effective delivery to the lung is difficult because of the complex branching structure of the airways. My study addresses this delivery problem by using exogenous surfactant, a pulmonary delivery vehicle capable of improving spreading of these peptides throughout the ...


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