3D Localization of Retrovirus Assembly in the Presence of Structured Background with Deep Learning.

Human immunodeficiency virus type 1 (HIV-1) particle assembly is driven by the Gag structural polyprotein and is a crucial step in the production of new virus particles. Elucidating the details of this process is necessary to fully understand the virus replication cycle. Real-time measurements of virus particle biogenesis in living cells have proved challenging, and most of our knowledge of this process to date has come from total internal fluorescence microscopy of labeled Gag at the bottom plasma membrane (PM) of adherent cells.

Identification of the GABARAP binding determinant in PI4K2A.

GABARAP is a member of the ATG8 family of ubiquitin-like autophagy related proteins. It was initially discovered as a facilitator of GABA-A receptor translocation to the plasma membrane and has since been shown to promote the intracellular transport of a variety of other proteins under non-autophagic conditions. We and others have shown that GABARAP interacts with the Type II phosphatidylinositol 4-kinase, PI4K2A, and that this interaction is important for autophagosome-lysosome fusion.

Analysis of Arc/Arg3.1 Oligomerization In Vitro and in Living Cells.

Arc (also known as Arg3.1) is an activity-dependent immediate early gene product enriched in neuronal dendrites. Arc plays essential roles in long-term potentiation, long-term depression, and synaptic scaling.

Statistical analysis of the autocorrelation function in fluorescence correlation spectroscopy.

Fluorescence correlation spectroscopy (FCS) is a powerful method to measure concentration, mobility, and stoichiometry in solution and in living cells, but quantitative analysis of FCS data remains challenging due to the correlated noise in the autocorrelation function (ACF) of FCS. We demonstrate here that least-squares fitting of the conventional ACF is incompatible with the χ goodness-of-fit test and systematically underestimates the true fit parameter uncertainty. To overcome this challenge, a simple method to fit the ACF is introduced that allows proper calculation of goodness-of-fit statistics and that provides more tightly constrained parameter estimates than the conventional least-squares fitting method, achieving the theoretical minimum uncertainty.

Autocorrelation function of finite-length data in fluorescence correlation spectroscopy.

The experimental autocorrelation function of fluorescence correlation spectroscopy calculated from finite-length data is a biased estimator of the theoretical correlation function. This study presents a new theoretical framework that explicitly accounts for the data length to allow for unbiased analysis of experimental autocorrelation functions. To validate our theory, we applied it to experiments and simulations of diffusion and characterized the accuracy and precision of the resulting parameter estimates.

Partitioning of ribonucleoprotein complexes from the cellular actin cortex.

The cell cortex plays a crucial role in cell mechanics, signaling, and development. However, little is known about the influence of the cortical meshwork on the spatial distribution of cytoplasmic biomolecules. Here, we describe a fluorescence microscopy method with the capacity to infer the intracellular distribution of labeled biomolecules with subresolution accuracy.

Molecular Determinants of Human T-cell Leukemia Virus Type 1 Gag Targeting to the Plasma Membrane for Assembly.

Assembly of human T-cell leukemia virus type 1 (HTLV-1) particles is initiated by the trafficking of virally encoded Gag polyproteins to the inner leaflet of the plasma membrane (PM). Gag-PM interactions are mediated by the matrix (MA) domain, which contains a myristoyl group (myr) and a basic patch formed by lysine and arginine residues. For many retroviruses, Gag-PM interactions are mediated by phosphatidylinositol 4,5-bisphosphate [PI(4,5)P]; however, previous studies suggested that HTLV-1 Gag-PM interactions and therefore virus assembly are less dependent on PI(4,5)P.

Gain-of-Function Properties of a Dynamin 2 Mutant Implicated in Charcot-Marie-Tooth Disease.

Mutations in the gene encoding dynamin 2 (DNM2), a GTPase that catalyzes membrane constriction and fission, are associated with two autosomal-dominant motor disorders, Charcot-Marie-Tooth disease (CMT) and centronuclear myopathy (CNM), which affect nerve and muscle, respectively. Many of these mutations affect the pleckstrin homology domain of DNM2, yet there is almost no overlap between the sets of mutations that cause CMT or CNM. A subset of CMT-linked mutations inhibit the interaction of DNM2 with phosphatidylinositol (4,5) bisphosphate, which is essential for DNM2 function in endocytosis.

Differentiating Luminal and Membrane-Associated Nuclear Envelope Proteins.

The nuclear envelope (NE) consists of two concentric nuclear membranes separated by the lumen, an ∼40-nm-wide fluid layer. NE proteins are implicated in important cellular processes ranging from gene expression to nuclear positioning. Although recent progress has been achieved in quantifying the assembly states of NE proteins in their native environment with fluorescence fluctuation spectroscopy, these studies raised questions regarding the association of NE proteins with nuclear membranes during the assembly process.

Time-shifted mean-segmented Q data of a luminal protein measured at the nuclear envelope by fluorescence fluctuation microscopy.

Fluorescence fluctuation microscopy is a widely used method to determine the mobility and oligomeric state of proteins in the live cell environment. Existing analysis methods rely on statistical evaluation of data segments with the implicit assumption that no significant signal fluctuations occur on the time scale of a data segment. Recent work on extending fluorescence fluctuation methods to the nuclear envelope of living cells identified a slow fluctuation process that is associated with the undulations of the nuclear membranes, which lead to intensity fluctuations due to local volume changes at the nuclear envelope.

Sensitive Detection of Protein Binding to the Plasma Membrane with Dual-Color Z-Scan Fluorescence.

Delicate and transitory protein engagement at the plasma membrane (PM) is crucial to a broad range of cellular functions, including cell motility, signal transduction, and virus replication. Here, we describe a dual-color (DC) extension of the fluorescence z-scan technique, which has proven successful for quantification of peripheral membrane protein binding to the PM in living cells. We demonstrate that the coexpression of a second, distinctly colored fluorescent protein provides a soluble reference species that delineates the extent of the cell cytoplasm and lowers the detection threshold of z-scan PM-binding measurements by an order of magnitude.

Identifying Heteroprotein Complexes in the Nuclear Envelope.

The nucleus is delineated by the nuclear envelope (NE), which is a double membrane barrier composed of the inner and outer nuclear membranes as well as a ∼40-nm wide lumen. In addition to its barrier function, the NE acts as a critical signaling node for a variety of cellular processes, which are mediated by protein complexes within this subcellular compartment. Although fluorescence fluctuation spectroscopy is a powerful tool for characterizing protein complexes in living cells, it was recently demonstrated that conventional fluorescence fluctuation spectroscopy methods are not suitable for applications in the NE because of the presence of slow nuclear membrane undulations.

Quantitative modeling of self-oligomerization of proteins in the nuclear envelope by fluorescence fluctuation analysis.

Analysis of fluorescence fluctuation data through the time-shifted mean-segmented Q (tsMSQ) analysis method has recently been shown to successfully identify protein oligomerization and mobility in the nuclear envelope by properly accounting for local volume fluctuations of the nuclear envelope within living cells. However, by its nature, tsMSQ produces correlated data which poses unique challenges for applying goodness of fit tests and obtaining parameter uncertainties from individual measurements. In this paper, we overcome these challenges by introducing bootstrap tsMSQ which involves randomly resampling the fluorescence intensity data to eliminate the correlations in the tsMSQ data.

Myristoylation-Dependent Palmitoylation of the Receptor Tyrosine Kinase Adaptor FRS2α.

An early step in signaling from activated receptor tyrosine kinases (RTKs) is the recruitment of cytosolic adaptor proteins to autophosphorylated tyrosines in the receptor cytoplasmic domains. Fibroblast growth factor receptor substrate 2α (FRS2α) associates via its phosphotyrosine-binding domain (PTB) to FGF receptors (FGFRs). Upon FGFR activation, FRS2α undergoes phosphorylation on multiple tyrosines, triggering recruitment of the adaptor Grb2 and the tyrosine phosphatase Shp2, resulting in stimulation of PI3K/AKT and MAPK signaling pathways.

Protein oligomerization and mobility within the nuclear envelope evaluated by the time-shifted mean-segmented Q factor.

Analysis of fluorescence fluctuation experiments by the mean-segmented Q (MSQ) method was recently used to successfully characterize the oligomeric state and mobility of proteins within the nuclear envelope (NE) of living cells. However, two significant shortcomings of MSQ were recognized. Non-ideal detector behavior due to dead-time and afterpulsing as well as the lack of error analysis currently limit the potential of MSQ.

Distinct Pathway of Human T-Cell Leukemia Virus Type 1 Gag Punctum Biogenesis Provides New Insights into Enveloped Virus Assembly.

The assembly of virus particles is a crucial aspect of virus spread. For retroviruses, the Gag polyprotein is the key driver for virus particle assembly. In order to produce progeny virus, once Gag is translated, it must translocate from the location in the cytoplasm where it is synthesized to the plasma membrane and form an oligomeric lattice that results in Gag puncta.

Investigating LINC Complex Protein Homo-oligomerization in the Nuclear Envelopes of Living Cells Using Fluorescence Fluctuation Spectroscopy.

Linkers of nucleoskeleton and cytoskeleton (LINC) complexes are conserved nuclear envelope (NE) spanning molecular bridges which mechanically integrate the nucleus with the cytoskeleton and mediate force transmission into the nucleoplasm. Despite their critical roles in fundamental cellular processes such as meiotic chromosome and nuclear positioning, the mechanism of LINC complex assembly in cells remains unclear. To begin to address this deficit, we recently developed z-scan fluorescence fluctuation spectroscopy (FFS) and brightness analysis as a method for quantifying the oligomeric states of fluorescent protein-tagged NE proteins including nesprins and SUN proteins.

Comobility of GABARAP and Phosphatidylinositol 4-Kinase 2A on Cytoplasmic Vesicles.

We previously reported that recruitment of the type IIA phosphatidylinositol 4-kinase (PI4K2A) to autophagosomes by GABARAP, a member of the Atg8 family of autophagy-related proteins, is important for autophagosome-lysosome fusion. Because both PI4K2A and GABARAP have also been implicated in the intracellular trafficking of plasma membrane receptors in the secretory/endocytic pathway, we characterized their interaction in cells under nonautophagic conditions. Fluorescence fluctuation spectroscopy measurements revealed that GABARAP exists predominantly as a cytosolic monomer in live cells, but is recruited to small cytoplasmic vesicles upon overexpression of PI4K2A.

Critical Role of the Human T-Cell Leukemia Virus Type 1 Capsid N-Terminal Domain for Gag-Gag Interactions and Virus Particle Assembly.

The retroviral Gag protein is the main structural protein responsible for virus particle assembly and release. Like human immunodeficiency virus type 1 (HIV-1) Gag, human T-cell leukemia virus type 1 (HTLV-1) has a structurally conserved capsid (CA) domain, including a β-hairpin turn and a centralized coiled-coil-like structure of six α helices in the CA amino-terminal domain (NTD), as well as four α-helices in the CA carboxy-terminal domain (CTD). CA drives Gag oligomerization, which is critical for both immature Gag lattice formation and particle production.

Fluorescence fluctuation spectroscopy reveals differential SUN protein oligomerization in living cells.

Linker-of-nucleoskeleton-and-cytoskeleton (LINC) complexes are conserved molecular bridges within the nuclear envelope that mediate mechanical force transmission into the nucleoplasm. The core of a LINC complex is formed by a transluminal interaction between the outer and inner nuclear membrane KASH and SUN proteins, respectively. Mammals encode six KASH proteins and five SUN proteins.

Perturbation of Human T-Cell Leukemia Virus Type 1 Particle Morphology by Differential Gag Co-Packaging.

Human T-cell leukemia virus type 1 (HTLV-1) is an important cancer-causing human retrovirus that has infected approximately 15 million individuals worldwide. Many aspects of HTLV-1 replication, including virus particle structure and assembly, are poorly understood. Group-specific antigen (Gag) proteins labeled at the carboxy terminus with a fluorophore protein have been used extensively as a surrogate for fluorescence studies of retroviral assembly.

Quantitative Brightness Analysis of Protein Oligomerization in the Nuclear Envelope.

Brightness analysis of fluorescence fluctuation experiments has been used to successfully measure the oligomeric state of proteins at the plasma membrane, in the nucleoplasm, and in the cytoplasm of living cells. Here we extend brightness analysis to the nuclear envelope (NE), a double membrane barrier separating the cytoplasm from the nucleoplasm. Results obtained by applying conventional brightness analysis to fluorescently tagged proteins within the NE exhibited an unusual concentration dependence.

Disparate Contributions of Human Retrovirus Capsid Subdomains to Gag-Gag Oligomerization, Virus Morphology, and Particle Biogenesis.

The capsid domain (CA) of the retroviral Gag protein is a primary determinant of Gag oligomerization, which is a critical step for immature Gag lattice formation and virus particle budding. Although the human immunodeficiency virus type 1 (HIV-1) CA carboxy-terminal domain (CTD) is essential for CA-CA interactions, the CA CTD has been suggested to be largely dispensable for human T-cell leukemia virus type 1 (HTLV-1) particle biogenesis. To more clearly define the roles of the HTLV-1 CA amino-terminal domain (NTD) and CA CTD in particle biogenesis, we generated and analyzed a panel of Gag proteins with chimeric HIV-1/HTLV-1 CA domains.

Association of Endophilin B1 with Cytoplasmic Vesicles.

Endophilins are SH3- and BAR domain-containing proteins implicated in membrane remodeling and vesicle formation. Endophilins A1 and A2 promote the budding of endocytic vesicles from the plasma membrane, whereas endophilin B1 has been implicated in vesicle budding from intracellular organelles, including the trans-Golgi network and late endosomes. We previously reported that endophilins A1 and A2 exist almost exclusively as soluble dimers in the cytosol.