Analysis of Protein Turnover by Quantitative SNAP-Based Pulse-Chase Imaging

Assessment of protein dynamics in living cells is crucial for understanding their biological properties and functions. The SNAP-tag, a self labeling suicide enzyme, presents a tool with unique features that can be adopted for determining protein dynamics in living cells. Here we present detailed protocols for the use of SNAP in fluorescent pulse-chase and quench-chase-pulse experiments. These time-slicing methods provide powerful tools to assay and quantify the fate and turnover rate of proteins of different ages. We cover advantages and pitfalls of SNAP-tagging in fixed- and live-cell studies and evaluate the recently developed fast-acting SNAPf variant. In addition, to facilitate the analysis of protein turnover datasets, we present an automated algorithm for spot recognition and quantification.

Cdk Activity Couples Epigenetic Centromere Inheritance to Cell Cycle Progression

Centromeres form the site of chromosome attachment to microtubules during mitosis. Identity of these loci is maintained epigenetically by nucleosomes containing the histone H3 variant CENP-A. Propagation of CENP-A chromatin is uncoupled from DNA replication initiating only during mitotic exit. We now demonstrate that inhibition of Cdk1 and Cdk2 activities is sufficient to trigger CENP-A assembly throughout the cell cycle in a manner dependent on the canonical CENP-A assembly machinery. We further show that the key CENP-A assembly factor Mis18BP1(HsKNL2) is phosphorylated in a cell cycle-dependent manner that controls its centromere localization during mitotic exit. These results strongly support a model in which the CENP-A assembly machinery is poised for activation throughout the cell cycle but kept in an inactive noncentromeric state by Cdk activity during S, G2, and M phases. Alleviation of this inhibition in G1 phase ensures tight coupling between DNA replication, cell division, and subsequent centromere maturation.

How two become one: HJURP dimerization drives CENP-A assembly

CENP‐A containing nucleosomes epigenetically specify centromere position on chromosomes. Deposition of CENP‐A into chromatin is mediated by HJURP, a specific CENP‐A chaperone. Paradoxically, HJURP binding sterically prevents dimerization of CENP‐A, which is critical to form functional centromeric nucleosomes. A recent publication in The EMBO Journal (Zasadzińska et al, 2013) demonstrates that HJURP itself dimerizes through a C‐terminal repeat region, which is essential for centromeric assembly of nascent CENP‐A.

University of Michigan Men's Basketball Team, 2004

Back Row: coach Tommy Amaker, director of basketball operations Kirsten Green, asst. coach Chuck Swenson, asst. coach Charles E. Ramsey, Lester Abram, Brent Petway, Amadou Ba, Chris Hunter, Courtney Sims, Graham Brown, John Andrews, asst. coach Andrew Moore, strength coach Jim Plocki, academic advisor Support Angie Heath, equip. mngr. Bob Bland.

Front Row: trainer Eric Broekhuizen, mngr. Michael Oh, Sherrod Harrell, Daniel Horton, Colin Dill, Bernard Robinson Jr., J.C. Mathis, Dion Harris, Dani Wohl, Manager Matt Duprey, admin. asst. Lucien St. Gerard.

University of Michigan Men's Basketball Team, 2004

Back Row: Lester Abram, Brent Petway, Amadou Ba, Chris Hunter, Courtney Sims, Graham Brown, and John Andrews.

Front Row: Sherrod Harrell, Daniel Horton, Colin Dill, Bernard Robinson Jr., J.C. Mathis, Dion Harris, and Dani Wohl.

University of Michigan Men's Basketball Team, 2004 NIT Champions

Back Row: Colin Dill, Brett Petway, Chris Hunter, Courtney Sims, Amadou Ba, Graham Brown, J.C. Mathis

Front Row:Dani Wohl, Daniel Horton, Sherrod Harrell, Lester Abram, John Andrews, Dion Harris, Ashtyn Bell

University of Michigan Men's Basketball Team, 2005

Back Row: head coach Tommy Amaker, director of basketball operations Kirsten Green, asst. coach Chuck Swenson, asst. coach Charles Ramsey, John Andrews, Brent Petway, Courtney Sims, Chris Hunter, Amadou Ba, Graham Brown, Ronald Coleman, asst. coach Andrew More academic advisor Angie Beck, strength coach Jim Plocki, equip. mngr. Bob Bland.

Front Row: administrative asst. Lucien St. Gerard, Dani Wohl, Dion Harris, Sherrod Harrell, J.C. Mathis, Lester Abram, Daniel Bell, sr. mngr. Matt Duprey.