drosophila-embryo-preparation-microinjection-for-live-cell-microscopy

Drosophila Embryo Preparation and Microinjection for Live Cell Microscopy Performed using an Automated High Content Analyzer

Modern approaches in quantitative live cell imaging have become an essential tool for exploring cell biology, by enabling the use of statistics and ...

San Francisco State University

Intracellular membrane networks including the endoplasmic reticulum (ER) and the Golgi apparatus experience dramatic reorganization upon entry into mitosis. However, the mechanisms driving these rearrangements and their importance for cell division are poorly understood. The GTPase Sar1 is a component of the secretory pathway and a key activator of anterograde transport of cargo from the ER to the Golgi. Here we show that Sar1 mutant proteins added to metaphase-arrested Xenopus laevis egg extracts cause dramatic effects on membrane organization. Live analysis of membrane structures in egg extract cytoplasm revealed a distinct network of sheets and tubules reflective of the organization of the ER in other systems. Addition of a constitutively active Sar1 GTPase mutant (H79G) increased membrane tubulation, while a dominant negative version Sar1 (T39N) impaired tubule organization. Although microtubule pelleting assays revealed that Sar1 associates with microtubules in the egg extract, and addition of Sar1 (H79G) mutant slightly destabilized spindle poles, bipolar spindle assembly was largely unaffected. Thus, spindles are stable to dramatic changes in mitotic membrane organization at metaphase, suggesting that mitotic membrane is not an upstream regulator of the mitotic spindle apparatus in Xenopus egg extracts.

Altering membrane topology with Sar1 does not impair spindle assembly in Xenopus egg extracts.

Mitotic cyclin-dependent kinase with their cyclin partners (cyclin:Cdks) are the master regulators of cell cycle progression responsible for regulating a host of activities during mitosis. Nuclear mitotic events, including chromosome condensation and segregation have been directly linked to Cdk activity. However, the regulation and timing of cytoplasmic mitotic events by cyclin:Cdks is poorly understood. In order to examine these mitotic cytoplasmic events, we looked at the dramatic changes in the endoplasmic reticulum (ER) during mitosis in the early Drosophila embryo. The dynamic changes of the ER can be arrested in an interphase state by inhibition of either DNA or protein synthesis. Here we show that this block can be alleviated by micro-injection of Cyclin A (CycA) in which defined mitotic ER clusters gathered at the spindle poles. Conversely, micro-injection of Cyclin B (CycB) did not affect spatial reorganization of the ER, suggesting CycA possesses the ability to initiate mitotic ER events in the cytoplasm. Additionally, RNAi-mediated simultaneous inhibition of all 3 mitotic cyclins (A, B and B3) blocked spatial reorganization of the ER. Our results suggest that mitotic ER reorganization events rely on CycA and that control and timing of nuclear and cytoplasmic events during mitosis may be defined by release of CycA from the nucleus as a consequence of breakdown of the nuclear envelope. 

Spatial reorganization of the endoplasmic reticulum during mitosis relies on mitotic kinase cyclin A in the early Drosophila embryo.

Mechanisms that regulate partitioning of the endoplasmic reticulum (ER) during cell division are largely unknown. Previous studies have mostly addressed ER partitioning in cultured cells, which may not recapitulate physiological processes that are critical in developing, intact tissues. We have addressed this by analysing ER partitioning in asymmetrically dividing stem cells, in which precise segregation of cellular components is essential for proper development and tissue architecture. We show that in Drosophila neural stem cells, called neuroblasts, the ER asymmetrically partitioned to centrosomes early in mitosis. This correlated closely with the asymmetric nucleation of astral microtubules (MTs) by centrosomes, suggesting that astral MT association may be required for ER partitioning by centrosomes. Consistent with this, the ER also associated with astral MTs in meiotic Drosophila spermatocytes and during syncytial embryonic divisions. Disruption of centrosomes in each of these cell types led to improper ER partitioning, demonstrating the critical role for centrosomes and associated astral MTs in this process. Importantly, we show that the ER also associated with astral MTs in cultured human cells, suggesting that this centrosome/astral MT-based partitioning mechanism is conserved across animal species. 

Proper symmetric and asymmetric endoplasmic reticulum partitioning requires astral microtubules.

Near-peer STEM Mentoring Offers Unexpected Benefits for Mentors from Traditionally Underrepresented Backgrounds.

Active-learning pedagogies have been repeatedly demonstrated to produce superior learning gains with large effect sizes compared with lecture-based pedagogies. Shifting large numbers of college science, technology, engineering, and mathematics (STEM) faculty to include any active learning in their teaching may retain and more effectively educate far more students than having a few faculty completely transform their teaching, but the extent to which STEM faculty are changing their teaching methods is unclear. Here, we describe the development and application of the machine-learning-derived algorithm Decibel Analysis for Research in Teaching (DART), which can analyze thousands of hours of STEM course audio recordings quickly, with minimal costs, and without need for human observers. DART analyzes the volume and variance of classroom recordings to predict the quantity of time spent on single voice (e.g., lecture), multiple voice (e.g., pair discussion), and no voice (e.g., clicker question thinking) activities. Applying DART to 1,486 recordings of class sessions from 67 courses, a total of 1,720 h of audio, revealed varied patterns of lecture (single voice) and nonlecture activity (multiple and no voice) use. We also found that there was significantly more use of multiple and no voice strategies in courses for STEM majors compared with courses for non-STEM majors, indicating that DART can be used to compare teaching strategies in different types of courses. Therefore, DART has the potential to systematically inventory the presence of active learning with ∼90% accuracy across thousands of courses in diverse settings with minimal effort.

Classroom sound can be used to classify teaching practices in college science courses.

Asymmetric cell division is the primary mechanism to generate cellular diversity, and it relies on the correct partitioning of cell fate determinants. However, the mechanism by which these determinants are delivered and positioned is poorly understood, and the upstream signal to initiate asymmetric cell division is unknown. Here we report that the endoplasmic reticulum (ER) is asymmetrically partitioned during mitosis in epithelial cells just before delamination and selection of a proneural cell fate in the early  embryo. At the start of gastrulation, the ER divides asymmetrically into a population of asynchronously dividing cells at the anterior end of the embryo. We found that this asymmetric division of the ER depends on the highly conserved ER membrane protein Jagunal (Jagn). RNA inhibition of  just before the start of gastrulation disrupts this asymmetric division of the ER. In addition, -deficient embryos display defects in apical-basal spindle orientation in delaminated embryonic neuroblasts. Our results describe a model in which an organelle is partitioned asymmetrically in an otherwise symmetrically dividing cell population just upstream of cell fate determination and updates previous models of spindle-based selection of cell fate during mitosis.

The endoplasmic reticulum is partitioned asymmetrically during mitosis before cell fate selection in proneuronal cells in the early  embryo.

Many efforts to improve science teaching in higher education focus on a few faculty members at an institution at a time, with limited published evidence on attempts to engage faculty across entire departments. We created a long-term, department-wide collaborative professional development program, Biology Faculty Explorations in Scientific Teaching (Biology FEST). Across 3 years of Biology FEST, 89% of the department's faculty completed a weeklong scientific teaching institute, and 83% of eligible instructors participated in additional semester-long follow-up programs. A semester after institute completion, the majority of Biology FEST alumni reported adding active learning to their courses. These instructor self-reports were corroborated by audio analysis of classroom noise and surveys of students in biology courses on the frequency of active-learning techniques used in classes taught by Biology FEST alumni and nonalumni. Three years after Biology FEST launched, faculty participants overwhelmingly reported that their teaching was positively affected. Unexpectedly, most respondents also believed that they had improved relationships with departmental colleagues and felt a greater sense of belonging to the department. Overall, our results indicate that biology department-wide collaborative efforts to develop scientific teaching skills can indeed attract large numbers of faculty, spark widespread change in teaching practices, and improve departmental relations.

Collectively Improving Our Teaching: Attempting Biology Department-wide Professional Development in Scientific Teaching.

Ataxin-2, a conserved RNA-binding protein, is implicated in the late-onset neurodegenerative disease Spinocerebellar ataxia type-2 (SCA2). SCA2 is characterized by shrunken dendritic arbors and torpedo-like axons within the Purkinje neurons of the cerebellum. Torpedo-like axons have been described to contain displaced endoplasmic reticulum (ER) in the periphery of the cell; however, the role of Ataxin-2 in mediating ER function in SCA2 is unclear. We utilized the Caenorhabditis elegans and Drosophila homologs of Ataxin-2 (ATX-2 and DAtx2, respectively) to determine the role of Ataxin-2 in ER function and dynamics in embryos and neurons. Loss of ATX-2 and DAtx2 resulted in collapse of the ER in dividing embryonic cells and germline, and ultrastructure analysis revealed unique spherical stacks of ER in mature oocytes and fragmented and truncated ER tubules in the embryo. ATX-2 and DAtx2 reside in puncta adjacent to the ER in both C. elegans and Drosophila embryos. Lastly, depletion of DAtx2 in cultured Drosophila neurons recapitulated the shrunken dendritic arbor phenotype of SCA2. ER morphology and dynamics were severely disrupted in these neurons. Taken together, we provide evidence that Ataxin-2 plays an evolutionary conserved role in ER dynamics and morphology in C. elegans and Drosophila embryos during development and in fly neurons, suggesting a possible SCA2 disease mechanism.

Conserved role for Ataxin-2 in mediating endoplasmic reticulum dynamics.

Instructor Talk-noncontent language used by instructors in classrooms-is a recently defined and promising variable for better understanding classroom dynamics. Having previously characterized the Instructor Talk framework within the context of a single course, we present here our results surrounding the applicability of the Instructor Talk framework to noncontent language used by instructors in novel course contexts. We analyzed Instructor Talk in eight additional biology courses in their entirety and in 61 biology courses using an emergent sampling strategy. We observed widespread use of Instructor Talk with variation in the amount and category type used. The vast majority of Instructor Talk could be characterized using the originally published Instructor Talk framework, suggesting the robustness of this framework. Additionally, a new form of Instructor Talk-Negatively Phrased Instructor Talk, language that may discourage students or distract from the learning process-was detected in these novel course contexts. Finally, the emergent sampling strategy described here may allow investigation of Instructor Talk in even larger numbers of courses across institutions and disciplines. Given its widespread use, potential influence on students in learning environments, and ability to be sampled, Instructor Talk may be a key variable to consider in future research on teaching and learning in higher education.

Investigating Instructor Talk in Novel Contexts: Widespread Use, Unexpected Categories, and an Emergent Sampling Strategy.

During mitosis, the structure of the Endoplasmic Reticulum (ER) displays a dramatic reorganization and remodeling, however, the mechanism driving these changes is poorly understood. Hairpin-containing ER transmembrane proteins that stabilize ER tubules have been identified as possible factors to promote these drastic changes in ER morphology. Recently, the Reticulon and REEP family of ER shaping proteins have been shown to heavily influence ER morphology by driving the formation of ER tubules, which are known for their close proximity with microtubules. Here, we examine the role of microtubules and other cytoskeletal factors in the dynamics of a Drosophila Reticulon, Reticulon-like 1 (Rtnl1), localization to spindle poles during mitosis in the early embryo. At prometaphase, Rtnl1 is enriched to spindle poles just prior to the ER retention motif KDEL, suggesting a possible recruitment role for Rtnl1 in the bulk localization of ER to spindle poles. Using image analysis-based methods and precise temporal injections of cytoskeletal inhibitors in the early syncytial Drosophila embryo, we show that microtubules are necessary for proper Rtnl1 localization to spindles during mitosis. Lastly, we show that astral microtubules, not microfilaments, are necessary for proper Rtnl1 localization to spindle poles, and is largely independent of the minus-end directed motor protein dynein. This work highlights the role of the microtubule cytoskeleton in Rtnl1 localization to spindles during mitosis and sheds light on a pathway towards inheritance of this major organelle.

Microtubules are necessary for proper Reticulon localization during mitosis.

Over the past decade, growing evidence has shown that there are many benefits to undergraduate students engaging in scientific research, including increased persistence in pursuing STEM careers and successful outcomes in graduate study. With these benefits in mind, there has been a significant push toward providing research opportunities for students in STEM majors. To address this need, an increasing number of undergraduate courses have been developed to provide students with research experiences in a class setting, also known as course-based undergraduate research experiences, or CUREs. Despite the growing success of these courses, a number of barriers remain that deter faculty from developing and implementing CUREs. Here, we will review the perceived challenges of developing a CURE and provide practical strategies to overcome these challenges.

Article	Year
Altering membrane topology with Sar1 does not impair spindle assembly in Xenopus egg extracts. Cytoskeleton (Hoboken, N.J.)\| PubMed ID: 22605651	2012
Spatial reorganization of the endoplasmic reticulum during mitosis relies on mitotic kinase cyclin A in the early Drosophila embryo. PloS one\| PubMed ID: 25689737	2015
Proper symmetric and asymmetric endoplasmic reticulum partitioning requires astral microtubules. Open biology\| PubMed ID: 26289801	2015
Near-peer STEM Mentoring Offers Unexpected Benefits for Mentors from Traditionally Underrepresented Backgrounds. Perspectives on undergraduate research and mentoring : PURM\| PubMed ID: 27668127	2015
Classroom sound can be used to classify teaching practices in college science courses. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 28265087	2017
The endoplasmic reticulum is partitioned asymmetrically during mitosis before cell fate selection in proneuronal cells in the early embryo. Molecular biology of the cell\| PubMed ID: 28381427	2017
Collectively Improving Our Teaching: Attempting Biology Department-wide Professional Development in Scientific Teaching. CBE life sciences education\| PubMed ID: 29326102	2018
Conserved role for Ataxin-2 in mediating endoplasmic reticulum dynamics. Traffic (Copenhagen, Denmark)\| PubMed ID: 30989774	2019
Investigating Instructor Talk in Novel Contexts: Widespread Use, Unexpected Categories, and an Emergent Sampling Strategy. CBE life sciences education\| PubMed ID: 31469624	2019
Microtubules are necessary for proper Reticulon localization during mitosis. PloS one\| PubMed ID: 31877164	2019
Fear of the CURE: A Beginner's Guide to Overcoming Barriers in Creating a Course-Based Undergraduate Research Experience. Journal of microbiology & biology education\| PubMed ID: 32528607	2020

Blake Riggs

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