Tau fibrils induce nanoscale membrane damage and nucleate cytosolic tau at lysosomes.

The prion-like spread of protein aggregates is a leading hypothesis for the propagation of neurofibrillary lesions in the brain, including the spread of tau inclusions associated with Alzheimer's disease. The mechanisms of cellular uptake of tau seeds and subsequent nucleated polymerization of cytosolic tau are major questions in the field, and the potential for coupling between the entry and nucleation mechanisms has been little explored. We found that in primary astrocytes and neurons, endocytosis of tau seeds leads to their accumulation in lysosomes. This in turn leads to lysosomal swelling, deacidification, and recruitment of ESCRT proteins, but not Galectin-3, to the lysosomal membrane. These observations are consistent with nanoscale damage of the lysosomal membrane. Live cell imaging and STORM superresolution microscopy further show that the nucleation of cytosolic tau occurs primarily at the lysosome membrane under these conditions. These data suggest that tau seeds escape from lysosomes via nanoscale damage rather than wholesale rupture and that nucleation of cytosolic tau commences as soon as tau fibril ends emerge from the lysosomal membrane.

Single-molecule phospholipase A2 becomes processive on melittin-induced membrane deformations.

While it is established that the topology of lipid membranes plays an important role in biochemical processes, few direct observations exist regarding how the membranes are actively restructured and its consequences on subsequent reactions. In this work, we investigated how the two major components of bee venom, melittin and phospholipase A2 (PLA2), achieve activation by such membrane remodeling. Their membrane-disrupting functions have been reported to increase when both are present, but the mechanism of this synergism had not been established. Using membrane reconstitution, we found that melittin can form large-scale membrane deformities upon which PLA2 activity is 25-fold higher. Tracking of single-molecule PLA2 revealed that its processive behavior on these deformities underlies the enhanced activity. These results show how melittin and PLA2 work synergistically to enhance the lytic effects of the bee venom. More broadly, they also demonstrate how the membrane topology may be actively altered to modulate cellular membrane-bound reactions.

Impact of Changing Distance-Learning Formats in Physical Therapy Professional Education.

AIMS: In early 2020, the COVID-19 pandemic caused a majority of higher education to shift content delivery formats to allow for social distancing to decrease spread of the virus. The purpose of this investigation was to discover physical therapy students' perceived impact from changing from a synchronous videoconferencing format to a more hybrid program format. METHODS: A qualitative case study design bounded by the time of switching formats due to the pandemic was chosen (March 2020 through September 2020). Physical therapy students participated in an agreement survey (n=38) and semi-structured interviews (n=12). Survey and interview data were coded and analyzed to form categories and themes of discovered perspectives. RESULTS: Students' perceived proficiency of hands-on skill was negatively affected. Cross-campus student and faculty interactions improved. Students felt changing formats would not result in detrimental effects on their overall learning nor on their career potential as physical therapists. DISCUSSION: Educators in entry-level professional physical therapy programs utilizing distance-education models should consider and adjust timing of hands-on skill instruction to match didactic content to encourage better connection and clinical application. Distance-learning educators should foster more interaction with students who may feel isolated. Interaction between distance-separated cohorts can reduce feelings of competition and inequality between campus locations and create improved learning communities.

Machine-learning-powered extraction of molecular diffusivity from single-molecule images for super-resolution mapping.

While critical to biological processes, molecular diffusion is difficult to quantify, and spatial mapping of local diffusivity is even more challenging. Here we report a machine-learning-enabled approach, pixels-to-diffusivity (Pix2D), to directly extract the diffusion coefficient D from single-molecule images, and consequently enable super-resolved D spatial mapping. Working with single-molecule images recorded at a fixed framerate under typical single-molecule localization microscopy (SMLM) conditions, Pix2D exploits the often undesired yet evident motion blur, i.e., the convolution of single-molecule motion trajectory during the frame recording time with the diffraction-limited point spread function (PSF) of the microscope. Whereas the stochastic nature of diffusion imprints diverse diffusion trajectories to different molecules diffusing at the same given D, we construct a convolutional neural network (CNN) model that takes a stack of single-molecule images as the input and evaluates a D-value as the output. We thus validate robust D evaluation and spatial mapping with simulated data, and with experimental data successfully characterize D differences for supported lipid bilayers of different compositions and resolve gel and fluidic phases at the nanoscale.

Tau fibrils induce nanoscale membrane damage and nucleate cytosolic tau at lysosomes.

The prion-like spread of protein aggregates is a leading hypothesis for the propagation of neurofibrillary lesions in the brain, including the spread of tau inclusions associated with Alzheimer's disease. The mechanisms of cellular uptake of tau seeds and subsequent nucleated polymerization of cytosolic tau are major questions in the field, and the potential for coupling between the entry and nucleation mechanisms has been little explored. We found that in primary astrocytes, endocytosis of tau seeds leads to their accumulation in lysosomes. This in turn leads to lysosomal swelling, deacidification and recruitment of ESCRT proteins, but not Galectin-3, to the lysosomal membrane. These observations are consistent with nanoscale damage of the lysosomal membrane. Using live cell and STORM, imaging, nucleation of cytosolic tau occurs primarily at the lysosome membrane under these conditions. These data suggest that tau seeds escape from lysosomes via nanoscale damage rather than wholesale rupture, and that nucleation of cytosolic tau commences as soon as tau fibril ends emerge from the lysosomal membrane.

Diffusion-based determination of protein homodimerization on reconstituted membrane surfaces.

The transient interactions between cellular components, particularly on membrane surfaces, are critical in the proper function of many biochemical reactions. For example, many signaling pathways involve dimerization, oligomerization, or other types of clustering of signaling proteins as a key step in the signaling cascade. However, it is often experimentally challenging to directly observe and characterize the molecular mechanisms such interactions-the greatest difficulty lies in the fact that living cells have an unknown number of background processes that may or may not participate in the molecular process of interest, and as a consequence, it is usually impossible to definitively correlate an observation to a well-defined cellular mechanism. One of the experimental methods that can quantitatively capture these interactions is through membrane reconstitution, whereby a lipid bilayer is fabricated to mimic the membrane environment, and the biological components of interest are systematically introduced, without unknown background processes. This configuration allows the extensive use of fluorescence techniques, particularly fluorescence fluctuation spectroscopy and single-molecule fluorescence microscopy. In this review, we describe how the equilibrium diffusion of two proteins, K-Ras4B and the PH domain of Bruton's tyrosine kinase (Btk), on fluid lipid membranes can be used to determine the kinetics of homodimerization reactions. [BMB Reports 2021; 54(3): 157-163].