Biology subjects

Cooley, A.

Publications and source records attributed to Cooley, A..

5 recordsLinked to original sources

bioRxiv2023

Unique interactions and functions of the mitochondrial small Tims in Trypanosoma brucei

Trypanosoma brucei is an early divergent parasitic protozoan that causes a fatal disease, African trypanosomiasis. T. brucei possesses a unique and essential translocase of the mitochondrial inner membrane, the TbTIM17 complex. TbTim17 associates with 6 small TbTims, (TbTim9, TbTim10, TbTim11, TbTim12, TbTim13, and TbTim8/13). However, the interaction pattern of the small TbTims with each other and TbTim17 are not clear. Here, we demonstrated by yeast two-hybrid (Y2H) analysis that all six small TbTims interact with each other, but stronger interactions were found among TbTim8/13, TbTim9, and TbTim10. Each of the small TbTims also interact directly with the C-terminal region of TbTim17. RNAi studies indicated that among all small TbTims, TbTim13 is most crucial to maintain the steady-state levels of the TbTIM17 complex. Co-immunoprecipitation analyses from T. brucei mitochondrial extracts also showed that TbTim10 has a stronger association with TbTim9 and TbTim8/13, but a weaker association with TbTim13, whereas TbTim13 has a stronger connection with TbTim17. Analysis of the small TbTim complexes by size exclusion chromatography revealed that each small TbTim, except TbTim13, is present in [~]70 kDa complexes, which could be heterohexameric forms of the small TbTims. However, TbTim13 is primarily present in the larger complex (>800 kDa) and co-fractionated with TbTim17. Altogether, our results demonstrated that TbTim13 is a part of the TbTIM complex and the smaller complexes of the small TbTims likely interact with the larger complex dynamically. Therefore, relative to other eukaryotes, the architecture and function of the small TbTim complexes are specific in T. brucei.

cell biology↗

bioRxiv2022

Dynamic states of cervical epithelia during pregnancy and epithelial barrier disruption

The cervical epithelium undergoes continuous changes in proliferation, differentiation, and function that are critical before pregnancy to ensure fertility and during pregnancy to provide a physical and immunoprotective barrier for pregnancy maintenance. Barrier disruption can lead to the ascension of pathogens that elicit inflammatory responses and preterm birth. Here, we identify cervical epithelial subtypes in nonpregnant, pregnant, and in-labor mice using single-cell transcriptome and spatial analysis. We identify heterogeneous subpopulations of epithelia displaying spatial and temporal specificity. Notably, two goblet cell subtypes with distinct transcriptional programs and mucosal networks were dominant in pregnancy. Untimely basal cell proliferation and goblet cells with diminished mucosal integrity characterize barrier dysfunction in mice lacking hyaluronan. These data demonstrate how the cervical epithelium undergoes continuous remodeling to maintain dynamic states of homeostasis in pregnancy and labor, and provide a framework to understand perturbations in epithelial health and host-microbe interactions that increase the risk of premature birth.

cell biology↗

bioRxiv2021

Finding a Balance: Characterizing Teaching and Research Anxieties in Biology Graduate Teaching Assistants (GTAs)

Graduate students in the United States are reporting increased anxiety, affecting their mental health and attrition in graduate programs. Yet we are only beginning to understand what contributes to graduate student anxiety. Biology Graduate Teaching Assistants (GTAs) have simultaneous roles as teachers, researchers, students, and employees, and factors associated with these tasks may contribute to anxieties in graduate school, particularly in relation to teaching and research responsibilities. To explore factors related to GTA teaching and research anxieties, and guided by social cognitive career theory, we interviewed 23 Biology GTAs at a research-intensive southeastern university. Thematic analysis of interview transcripts revealed five major factors related to GTA anxieties: negative impact on self, negative impact on others, lack of self-efficacy, role tension, and personal anxieties. Lack of self-efficacy was most prevalent for research anxieties, compared to teaching anxieties, where the impact on others (e.g. students) was most prevalent. In research contexts, GTAs with academic career aspirations expressed less anxiety about role tensions compared to GTAs with non-academic career goals. By investigating GTA anxieties, this work can inform professional development or mental health interventions for GTAs and encourage greater awareness and dialogue about mental health issues in academia.

scientific communication and education↗

bioRxiv2021

Trypanosoma brucei Tim50 Plays a Critical Role in Cell Cycle Regulation and Parasite Infectivity

Tim50 is a receptor subunit of the preprotein-translocase of the mitochondrial inner membrane, TIM23. Trypanosoma brucei, the infective agent for African trypanosomiasis, possesses a homologue of Tim50 (TbTim50) with a pair of characteristic DXDX(T/V) phosphatase signature motifs. Here, we demonstrated that besides its protein phosphatase activity, the recombinant TbTim50 binds and hydrolyzes phosphatidic acid in a concentration-dependent manner. In silico structural homology models identify the putative binding interfaces that may accommodate different phospho-substrates. Interestingly, TbTim50 depletion in the bloodstream form (BF) of T. brucei reduced cardiolipin (CL) levels and decreased mitochondrial membrane potential ({Delta}{Psi}). TbTim50 knockdown (KD) also reduced the population of G2 phase and increased G1 phase; thus, BF cell growth was reduced. Confocal and electron microscopy revealed a defect in regulation of kinetoplast (kDNA) replication due to TbTim50 KD. Depletion of TbTim50 increased the levels of AMPK phosphorylation, and parasite morphology was changed to stumpy-like with upregulation of few stumpy marker gene expressions. Importantly, we observed that TbTim50-depleted parasites were unable to establish infection in mice and rats. Proteomics analysis showed reductions of the translation factors, flagellar transport proteins, and many proteasomal subunits, including the mitochondrial HslVU that is known to play a role in kDNA replication. Reduction of the level of HslV in TbTim50 KD cells was further validated by immunoblot analysis. Altogether, our results showed that TbTim50 is essential for mitochondrial function, regulation of kDNA replication, and cell cycle in the BF. Therefore, TbTim50 is an important target for structure-based drug design to combat African trypanosomiasis. ImportanceAfrican trypanosomiasis, a neglected tropical disease caused by parasitic protozoan Trypanosoma brucei, is transmitted by the tsetse fly prevalent in sub-Saharan Africa. During its digenetic life cycle, T. brucei undergoes multiple developmental changes to adapt in different environments. T. brucei BF, dwelling in mammalian blood, generates ATP from glycolysis and hydrolyzes ATP in mitochondria for inner membrane potential. We found that TbTim50, a HAD-family phosphatase, is critical for T. brucei BF survival in vitro and in vivo. Depletion of TbTim50 in BF reduced CL levels and mitochondrial {Delta}{Psi} and caused a detrimental effect on many cellular functions. Cells accumulated in G1-S phase, and kinetoplast was over-replicated due to depletion of mitochondrial proteasomes, HslVU, a master-regulator of kDNA replication. Cell growth inhibition was accompanied by changes in morphology, AMPK phosphorylation, and upregulation of stumpy-specific gene expression. TbTim50 is essential for T. brucei survival and an important T. brucei therapeutic target.

cell biology↗

bioRxiv2018

Frequent sgRNA-barcode Recombination in Single-cell Perturbation Assays

Simultaneously detecting CRISPR-based perturbations and induced transcriptional changes in the same cell is a powerful approach to unraveling genome function. Several lentiviral approaches have been developed, some of which rely on the detection of distally located genetic barcodes as an indirect proxy of sgRNA identity. Since barcodes are often several kilobases from their corresponding sgRNAs, viral recombination-mediated swapping of barcodes and sgRNAs is feasible. Using a self-circularization-based sgRNA-barcode library preparation protocol, we estimate the recombination rate to be ~50% and we trace this phenomenon to the pooled viral packaging step. Recombination is random, and decreases the signal-to-noise ratio of the assay. Our results suggest that alternative approaches can increase the throughput and sensitivity of single-cell perturbation assays.

genomics↗