BAG5 regulates HSPA8-mediated protein folding required for sperm head-tail coupling apparatus assembly.

Teratozoospermia is a significant cause of male infertility, but the pathogenic mechanism of acephalic spermatozoa syndrome (ASS), one of the most severe teratozoospermia, remains elusive. We previously reported Spermatogenesis Associated 6 (SPATA6) as the component of the sperm head-tail coupling apparatus (HTCA) required for normal assembly of the sperm head-tail conjunction, but the underlying molecular mechanism has not been explored. Here, we find that the co-chaperone protein BAG5, expressed in step 9-16 spermatids, is essential for sperm HTCA assembly. BAG5-deficient male mice show abnormal assembly of HTCA, leading to ASS and male infertility, phenocopying SPATA6-deficient mice. In vivo and in vitro experiments demonstrate that SPATA6, cargo transport-related myosin proteins (MYO5A and MYL6) and dynein proteins (DYNLT1, DCTN1, and DNAL1) are misfolded upon BAG5 depletion. Mechanistically, we find that BAG5 forms a complex with HSPA8 and promotes the folding of SPATA6 by enhancing HSPA8's affinity for substrate proteins. Collectively, our findings reveal a novel protein-regulated network in sperm formation in which BAG5 governs the assembly of the HTCA by activating the protein-folding function of HSPA8.

PPARγ phase separates with RXRα at PPREs to regulate target gene expression.

Peroxisome proliferator-activated receptor (PPAR)-γ is a key transcription activator controlling adipogenesis and lipid metabolism. PPARγ binds PPAR response elements (PPREs) as the obligate heterodimer with retinoid X receptor (RXR) α, but exactly how PPARγ orchestrates the transcriptional response is unknown. This study demonstrates that PPARγ forms phase-separated droplets in vitro and solid-like nuclear condensates in cell, which is intriguingly mediated by its DNA binding domain characterized by the zinc finger motif. Furthermore, PPARγ forms nuclear condensates at PPREs sites through phase separation to compartmentalize its heterodimer partner RXRα to initiate PPARγ-specific transcriptional activation. Finally, using an optogenetic approach, the enforced formation of PPARγ/RXRα condensates leads to preferential enrichment at PPREs sites and significantly promotes the expression of PPARγ target genes. These results define a novel mechanism by which PPARγ engages the phase separation principles for efficient and specific transcriptional activation.

Neutralizing Antibodies to SARS-CoV-2 Selected from a Human Antibody Library Constructed Decades Ago.

Combinatorial antibody libraries not only effectively reduce antibody discovery to a numbers game, but enable documentation of the history of antibody responses in an individual. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has prompted a wider application of this technology to meet the public health challenge of pandemic threats in the modern era. Herein, a combinatorial human antibody library constructed 20 years before the coronavirus disease 2019 (COVID-19) pandemic is used to discover three highly potent antibodies that selectively bind SARS-CoV-2 spike protein and neutralize authentic SARS-CoV-2 virus. Compared to neutralizing antibodies from COVID-19 patients with generally low somatic hypermutation (SHM), these three antibodies contain over 13-22 SHMs, many of which are involved in specific interactions in their crystal structures with SARS-CoV-2 spike receptor binding domain. The identification of these somatically mutated antibodies in a pre-pandemic library raises intriguing questions about the origin and evolution of these antibodies with respect to their reactivity with SARS-CoV-2.

SARS-CoV-2 nucleocapsid protein phase separates with G3BPs to disassemble stress granules and facilitate viral production.

A key to tackling the coronavirus disease 2019 (COVID-19) pandemic is to understand how severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) manages to outsmart host antiviral defense mechanisms. Stress granules (SGs), which are assembled during viral infection and function to sequester host and viral mRNAs and proteins, are part of the antiviral responses. Here, we show that the SARS-CoV-2 nucleocapsid (N) protein, an RNA binding protein essential for viral production, interacted with Ras-GTPase-activating protein SH3-domain-binding protein (G3BP) and disrupted SG assembly, both of which require intrinsically disordered region1 (IDR1) in N protein. The N protein partitioned into SGs through liquid-liquid phase separation with G3BP, and blocked the interaction of G3BP1 with other SG-related proteins. Moreover, the N protein domains important for phase separation with G3BP and SG disassembly were required for SARS-CoV-2 viral production. We propose that N protein-mediated SG disassembly is crucial for SARS-CoV-2 production.

Note: Microelectrode-shielding tip for scanning probe electron energy spectroscopy.

We report a novel microelectrode-shielding tip (ME tip) for scanning probe electron energy spectroscopy (SPEES). The shielding effect of this tip is studied through comparing the detection efficiency with the normal tip by both experiment and simulation. The results show that the backscattering count rate detected by the SPEES instrument using the normal tip begins to decrease as the tip approaches to the sample surface within 21 µm, while that using the ME tip only starts to drop off within 1 µm. This indicates that the electron energy spectra can be measured with the ME tip at a much closer tip-sample distance. Furthermore, it is also demonstrated that the ME tip can be used to obtain topography of the sample surface in situ simultaneously.

Nonlinear inelastic electron scattering from Au nanostructures induced by localized surface plasmon resonance.

Nonlinear electron scattering is a recently-discovered physical process observed during the localized plasmonic excitation of Ag nanostructures on graphite surface. In the present work, nonlinear electron scattering phenomena is experimentally verified on Au nanostructures by measuring inelastic scattering of electrons field-emitted from tungsten tip. The relative intensity of the electron-energy-loss peak associated with the plasmonic excitation of Au shows again to increase nonlinearly with the electric field generated by the tip-sample bias, demonstrating the generality of nonlinear electron scattering process in plasmonic system. Compared to the nonlinear electron scattering phenomena observed on Ag nanostructures, the nonlinear term for Au nanostructures is about 1 to 2 orders of magnitude smaller, which is in consistent with the field enhancement factor of Au and Ag nanostructures from both the surface-enhanced Raman spectroscopy experiments and the theoretical calculations.

Note: Electron energy spectroscopy mapping of surface with scanning tunneling microscope.

We report a novel scanning probe electron energy spectrometer (SPEES) which combines a double toroidal analyzer with a scanning tunneling microscope to achieve both topography imaging and electron energy spectroscopy mapping of surface in situ. The spatial resolution of spectroscopy mapping is determined to be better than 0.7 ± 0.2 µm at a tip sample distance of 7 µm. Meanwhile, the size of the field emission electron beam spot on the surface is also measured, and is about 3.6 ± 0.8 µm in diameter. This unambiguously demonstrates that the spatial resolution of SPEES technique can be much better than the size of the incident electron beam.