Advancements and Practical Considerations for Biophysical Research: Navigating the Challenges and Future of Super-resolution Microscopy.

The introduction of super-resolution microscopy (SRM) has significantly advanced our understanding of cellular and molecular dynamics, offering a detailed view previously beyond our reach. Implementing SRM in biophysical research, however, presents numerous challenges. This review addresses the crucial aspects of utilizing SRM effectively, from selecting appropriate fluorophores and preparing samples to analyzing complex data sets. We explore recent technological advancements and methodological improvements that enhance the capabilities of SRM. Emphasizing the integration of SRM with other analytical methods, we aim to overcome inherent limitations and expand the scope of biological insights achievable. By providing a comprehensive guide for choosing the most suitable SRM methods based on specific research objectives, we aim to empower researchers to explore complex biological processes with enhanced precision and clarity, thereby advancing the frontiers of biophysical research.

Generation of a homozygous knock-in human embryonic stem cell line expressing mEos4b-tagged CTR1.

Copper transporter 1 (CTR1) is the major membrane protein responsible for cellular copper (Cu) uptake and mediates cellular copper homeostasis. To elucidate CTR1's behavior using imaging approaches, we generated a homozygous knock-in human embryonic stem cell (hESC) clone expressing photoconvertible fluorescence protein mEos4b-tagged endogenous CTR1 using CRISPR-Cas9 mediated homologous recombination. The engineered cells express functional CTR1-mEos4b fusion and have normal stem cell morphology. They remain pluripotent and can be differentiated into all three germ layers in vitro. This resource allows the study of CTR1 at an endogenous level in different cellular contexts using microscopy.

Crossroads between membrane trafficking machinery and copper homeostasis in the nerve system.

Imbalanced copper homeostasis and perturbation of membrane trafficking are two common symptoms that have been associated with the pathogenesis of neurodegenerative and neurodevelopmental diseases. Accumulating evidence from biophysical, cellular and in vivo studies suggest that membrane trafficking orchestrates both copper homeostasis and neural functions-however, a systematic review of how copper homeostasis and membrane trafficking interplays in neurons remains lacking. Here, we summarize current knowledge of the general trafficking itineraries for copper transporters and highlight several critical membrane trafficking regulators in maintaining copper homeostasis. We discuss how membrane trafficking regulators may alter copper transporter distribution in different membrane compartments to regulate intracellular copper homeostasis. Using Parkinson's disease and MEDNIK as examples, we further elaborate how misregulated trafficking regulators may interplay parallelly or synergistically with copper dyshomeostasis in devastating pathogenesis in neurodegenerative diseases. Finally, we explore multiple unsolved questions and highlight the existing challenges to understand how copper homeostasis is modulated through membrane trafficking.

Generation of a homozygous knock-in human embryonic stem cell line expressing SNAP-tagged SOD1.

Superoxide Dismutase 1 (SOD1) is an antioxidant enzyme that protects the cells from radical oxygen species. To study the behavior of endogenous SOD1 under a microscope, we genetically modified H1 human embryonic stem cells (hESCs) to express SOD1 fused with a SNAP-tag, a protein tag that can be covalently labeled with a variety of synthetic probes. The engineered homozygous clone expressing SOD1-SNAP fusion proteins has normal stem cell morphology and karyotype, expresses pluripotency markers, and can be differentiated into all three germ layers in vitro, providing a versatile platform for imaging-based studies of SOD1.

Facilitated Unbinding via Multivalency-Enabled Ternary Complexes: New Paradigm for Protein-DNA Interactions.

Dynamic protein-DNA interactions constitute highly robust cellular machineries to fulfill cellular functions. A vast number of studies have focused on how DNA-binding proteins search for and interact with their target DNA segments and on what cellular cues can regulate protein binding, for which protein concentration is a most obvious one. In contrast, how protein unbinding could be regulated by protein concentration has evaded attention because protein unbinding from DNA is typically a unimolecular reaction and thus concentration independent. Recent single-molecule studies from multiple research groups have uncovered that protein concentration can facilitate the unbinding of DNA-bound proteins, revealing regulation of protein unbinding as another mechanistic paradigm for gene regulation. In this Account, we review these recent in vitro and in vivo single-molecule experiments that uncovered the concentration-facilitated protein unbinding by multiple types of DNA-binding proteins, including sequence-nonspecific DNA-binding proteins (e.g., nucleoid-associated proteins, NAP), sequence-specific DNA-binding proteins (e.g., metal-responsive transcription regulators CueR and ZntR), sequence-neutral single-stranded DNA-binding proteins (e.g., Replication protein A, RPA), and DNA polymerases. For the in vitro experiments, Marko's group investigated the exchange of GFP-tagged DNA-bound NAPs with nontagged NAPs in solution of increasing concentration using single-molecule magnetic-tweezers fluorescence microscopy. The faster fluorescence intensity decrease with higher nontagged NAP concentrations suggests that DNA-bound NAPs undergo faster exchange with higher free NAP concentrations. Chen's group used single-molecule fluorescence resonance energy transfer measurements to study the unbinding of CueR from its cognate oligomeric DNA. The average microscopic dwell times of DNA-bound states become shorter with increasing CueR concentrations in the surroundings, demonstrating that free CueR proteins can facilitate the unbinding of the incumbent one on DNA through either assisted dissociation or direct substitution. Greene's group studied the unbinding of RPAs from single-stranded DNA using total internal reflection fluorescence microscopy and DNA curtain techniques. The fluorescence intensity versus time traces show faster decay with higher wild-type RPA concentrations, indicating that DNA-bound RPAs can undergo a concentration-facilitated exchange when encountering excess free RPA. van Oijen's group investigated the leading/lagging-strand polymerase exchange events in the bacteriophage T7 and E. coli replication systems using a combination of single-molecule fluorescence microscopy and DNA-flow-stretching assay. The processivity was observed to have larger decrease when the concentration of the Y526F polymerase mutant increases, indicating that the unbinding of the polymerase is also concentration-dependent. Using stroboscopic imaging and single-molecule tracking, Chen's group further advanced their study into living bacterial cells. They found CueR, as well as its homologue ZntR, shows concentration-enhanced unbinding from its DNA-binding site in vivo. Mechanistic consensus has emerged from these in vitro and in vivo single-molecule studies that encompass a range of proteins with distinct biological functions. It involves multivalent contacts between protein and DNA. The multivalency enables the formation of ternary complexes as intermediates, which subsequently give rise to concentration-enhanced protein unbinding. As multivalent contacts are ubiquitous among DNA-interacting proteins, this multivalency-enabled facilitated unbinding mechanism thus provides a potentially general mechanistic paradigm in regulating protein-DNA interactions.

Neural cell proliferation and survival in the hippocampus of adult CaV 2.1 calcium ion channel mutant mice.

Tottering mutant mice carry a mutation in the pore-forming subunit (α1A) of CaV2.1 (P/Q-type) voltage-gated calcium ion (Ca2+) channels resulting in reduced neuronal Ca2+ current density. We assessed male tottering mice for spatial learning using the Morris water maze. Tottering mice performed worse than wild type mice, suggesting abnormal hippocampal function. Because Ca2+ influx via voltage-dependent Ca2+ channels regulates neuronal survival and function, we assessed hippocampus volume and cell density using hematoxylin and eosin stained serial sections. Adult hippocampal neurogenesis was assessed using 5-bromo-2'-deoxyuridine (BrdU) labeling with fluorescent immunohistochemistry (IHC) and proliferating cell nuclear antigen (PCNA) with diaminobenzidine IHC. We double-labeled neurons using fluorescence IHC with BrdU-neuronal nuclei (Neu-N) or double labeling of astrocytes using BrdU-glial fibrillary protein, respectively, to assess cell proliferation and survival. We assessed numbers of dying cells using fluoro-Jade histochemistry. Decreased hippocampal volume, increased dentate hilar and hippocampal CA1 cell densities were observed in tottering mice compared to wild type mice. Cell proliferation was increased in the hilus and CA2 region of tottering mice compared to wild type mice. Dendritic intersections in Sholl analysis were decreased for tottering mouse CA1 pyramidal neurons compared to wild type mice. The increased regional cell density coincides with increases in cell proliferation in similar, non-neurogenic areas of the hippocampus of tottering mice. Thus, hippocampal alterations observed in adult tottering mice appear to result from changes in neuronal morphology and proliferation in non-neurogenic areas of the hippocampus, and less through altered adult hippocampal neurogenesis or cell death.