The spindle assembly checkpoint (SAC) is critical for sensing defective microtubule–kinetochore attachments and tension across the kinetochore and functions to arrest cells in prometaphase to allow time to repair any errors before proceeding into anaphase. Dysregulation of the SAC leads to chromosome segregation errors that have been linked to human diseases like cancer. Although much has been learned about the composition of the SAC and the factors that regulate its activity, the proximity associations of core SAC components have not been explored in a systematic manner. Here, we have taken a BioID2-proximity-labeling proteomic approach to define the proximity protein environment for each of the five core SAC proteins BUB1, BUB3, BUBR1, MAD1L1, and MAD2L1 in mitotic-enriched populations of cells where the SAC is active. These five protein association maps were integrated to generate a SAC proximity protein network that contains multiple layers of information related to core SAC protein complexes, protein–protein interactions, and proximity associations. Our analysis validated many known SAC complexes and protein–protein interactions. Additionally, it uncovered new protein associations, including the ELYS–MAD1L1 interaction that we have validated, which lend insight into the functioning of core SAC proteins and highlight future areas of investigation to better understand the SAC.

Disulfide-linked bioconjugates allow the delivery of pharmacologically active or other cargo to specific tissues in a redox-sensitive fashion. However, an understanding of the kinetics, subcellular distribution, and mechanism of disulfide cleavage in such bioconjugates is generally lacking. Here, we report a modular disulfide-linked TAMRA-BODIPY based FRET probe that can be readily synthesized, modified, and conjugated to a cysteine-containing biomolecule to enable real-time monitoring of disulfide cleavage during receptor-mediated endocytosis in cells. We demonstrate the utility of this probe to study disulfide reduction during HER2 receptor-mediated uptake of a Cys-engineered anti-HER2 THIOMAB antibody. We found that introduction of positive, but not negative, charges in the probe improved retention of the BODIPY catabolite. This permitted the observation of significant disulfide cleavage in endosomes or lysosomes on par with proteolytic cleavage of a similarly charged valine–citrulline peptide-based probe. In general, the FRET probe we describe should enable real-time cellular monitoring of disulfide cleavage in other targeted delivery systems for mechanistic or diagnostic applications. Furthermore, modifications to the released BODIPY moiety permit evaluation of physicochemical properties that govern lysosomal egress or retention, which may have implications for the development of next-generation antibody–drug conjugates.

SUMMARYHuman cell division is a highly regulated process that relies on the accurate capture and movement of chromosomes to the metaphase plate. Errors in the fidelity of chromosome congression and alignment can lead to improper chromosome segregation, which is correlated with aneuploidy and tumorigenesis. Here we show that the dual specificity phosphatase DUSP7 is important for regulating chromosome alignment. DUSP7 bound to ERK2 and regulated the abundance of active phospho-ERK2 through its phosphatase activity. Overexpression of DUSP7, but not catalytic dead mutants, led to a marked decrease in phopho-ERK2 and mitotic chromosome misalignment, while knockdown of DUSP7 also led to defective chromosome congression that resulted in a prolonged mitosis. Consistently, chemical inhibition of the MEK kinase that phosphorylates ERK2 or ERK2 itself led to chromosome alignment defects. Our results support a model where MEK phosphorylation and DUSP7 dephosphorylation regulate the levels of active phospho-ERK2 to promote proper cell division.

Myosins are ATP-dependent actin-based molecular motors critical for diverse cellular processes like intracellular trafficking, cell motility, and cell invasion. During cell division, myosin MYO10 is important for proper mitotic spindle assembly, the anchoring of the spindle to the cortex, and positioning of the spindle to the cell mid-plane. However, myosins are regulated by myosin regulatory light chains (RLCs), and whether RLCs are important for cell division has remained unexplored. Here, we have determined that the previously uncharacterized myosin RLC Myl5 associates with the mitotic spindle and is required for cell division. We show that Myl5 localizes to the leading edge and filopodia during interphase and to mitotic spindle poles and spindle microtubules during early mitosis. Importantly, depletion of Myl5 led to defects in mitotic spindle assembly, chromosome congression, and chromosome segregation and to a slower transition through mitosis. Furthermore, Myl5 bound to MYO10 in vitro and co-localized with MYO10 at the spindle poles. These results suggest that Myl5 is important for cell division and that it may be performing its function through MYO10.

Targeting the leukemia proliferation cycle has been a successful approach to developing antileukemic therapies. However, drug screening efforts to identify novel antileukemic agents have been hampered by the lack of a suitable high-throughput screening platform for suspension cells that does not rely on flow cytometry analyses. We report the development of a novel leukemia cell-based high-throughput chemical screening platform for the discovery of cell cycle phase specific inhibitors that utilizes chemical cell cycle profiling. We have used this approach to analyze the cell cycle response of acute lymphoblastic leukemia CCRF-CEM cells to each of 181,420 drug-like compounds. This approach yielded cell cycle phase specific inhibitors of leukemia cell proliferation. Further analyses of the top G2-phase and M-phase inhibitors identified the leukemia specific inhibitor 1 (Leusin-1). Leusin-1 arrests cells in G2-phase and triggers an apoptotic cell death. Most importantly, Leusin-1 was more active in acute lymphoblastic leukemia cells than other types of leukemias, non-blood cancers, or normal cells and represents a lead molecule for developing antileukemic drugs.

Somatic mutations that perturb Parkin ubiquitin ligase activity and the misregulation of iron homeostasis have both been linked to Parkinson’s disease. Lactotransferrin (LTF) is a member of the family of transferrin iron binding proteins that regulate iron homeostasis, and increased levels of LTF and its receptor have been observed in neurodegenerative disorders like Parkinson’s disease. Here, we report that Parkin binds to LTF and ubiquitylates LTF to influence iron homeostasis. Parkin-dependent ubiquitylation of LTF occurred most often on lysines (K) 182 and 649. Substitution of K182 or K649 with alanine (K182A or K649A, respectively) led to a decrease in the level of LTF ubiquitylation, and substitution at both sites led to a major decrease in the level of LTF ubiquitylation. Importantly, Parkin-mediated ubiquitylation of LTF was critical for regulating intracellular iron levels as overexpression of LTF ubiquitylation site point mutants (K649A or K182A/K649A) led to an increase in intracellular iron levels measured by ICP-MS/MS. Consistently, RNAi-mediated depletion of Parkin led to an increase in intracellular iron levels in contrast to overexpression of Parkin that led to a decrease in intracellular iron levels. Together, these results indicate that Parkin binds to and ubiquitylates LTF to regulate intracellular iron levels. These results expand our understanding of the cellular processes that are perturbed when Parkin activity is disrupted and more broadly the mechanisms that contribute to Parkinson’s disease.

Bisphenol A (BPA) is a known endocrine disruptor and potential carcinogen found in polycarbonate plastic products, food packaging, and drinking water supplies. A quick and affordable detector, such as a whole‐cell biosensor that measures the presence of BPA, would greatly benefit safety and health. We set out to develop such a method by using riboswitch‐based biosensors. Riboswitches are regions of mRNAs that control downstream gene expression and can be used as genetic sensors to create whole‐cell biosensors. Although there are no naturally occurring BPA‐riboswitches, we reasoned that we could use the power of genetic selections to generate one. Starting from a known riboswitch that regulates expression of the downstream selection marker tetA linked to the reporter gene gfp, we used PCR or Gibson Assembly to replace the aptamer domain of the switch with 40 random bases, generating plasmid libraries. We then transformed Escherichia coli with these plasmids such that each individual bacterium harbored a unique member of the library. Libraries made by Gibson Assembly resulted in 108 unique members, while those made by PCR were generally on the order of 106 members. With libraries in hand, the dualistic nature of TetA allowed us to apply dual genetic selection to identify riboswitches that express tetA‐gfp only in response to BPA. TetA is a transporter protein that pumps the antibiotic tetracycline out of the cell, rendering cells tetracycline resistant. TetA also allows toxic metals, such as Ni2+, to enter the cell, inhibiting growth. The E. coli library was grown in the presence of BPA and tetracycline. Surviving cells were switched to medium with Ni2+ and no BPA. Only those cells that express tetA‐gfp exclusively in the presence of BPA were expected to survive both positive and negative selection. We succeeded in isolating unique riboswitches that converged to single sequences from starting libraries by bringing each library through three rounds of selection with varying tetracycline and Ni2+ conditions. Ultimately, our hits proved largely unsuccessful in producing a significant increase in fluorescence when incubated with BPA for either 6 or 16 hours compared to no‐ligand controls. In order to improve the ON/OFF ratio of our riboswitches, we integrated fluorescence‐activated cell sorting into our riboswitch‐development platform, allowing us to both select for survival through dual genetic selection and screen for maximum or minimum fluorescence depending on presence or absence of the ligand of interest. Further studies will include optimizing the obtained riboswitches by mutagenesis and directed evolution.Support or Funding InformationResearch funding is provided by the NSF.

Human cell division is a highly regulated process that relies on the accurate capture and movement of chromosomes to the metaphase plate. Errors in the fidelity of chromosome congression and alignment can lead to improper chromosome segregation, which is correlated with aneuploidy and tumorigenesis. These processes are known to be regulated by extracellular signal-regulated kinase 2 (ERK2) in other species, but the role of ERK2 in mitosis in mammals remains unclear. Here, we have identified the dual-specificity phosphatase 7 (DUSP7), known to display selectivity for ERK2, as important in regulating chromosome alignment. During mitosis, DUSP7 bound to ERK2 and regulated the abundance of active phospho-ERK2 through its phosphatase activity. Overexpression of DUSP7, but not catalytically inactive mutants, led to a decrease in the levels of phospho-ERK2 and mitotic chromosome misalignment, while knockdown of DUSP7 also led to defective chromosome congression that resulted in a prolonged mitosis. Consistently, knockdown or chemical inhibition of ERK2 or chemical inhibition of the MEK kinase that phosphorylates ERK2 led to chromosome alignment defects. Our results support a model wherein MEK-mediated phosphorylation and DUSP7-mediated dephosphorylation regulate the levels of active phospho-ERK2 to promote proper cell division.