Vickie J. LaMorte

UNC-51/ATG1 kinase regulates axonal transport by mediating motor–cargo assembly

Axonal transport mediated by microtubule-dependent motors is vital for neuronal function and viability. Selective sets of cargoes, including macromolecules and organelles, are transported long range along axons to specific destinations. Despite intensive studies focusing on the motor machinery, the regulatory mechanisms that control motor-cargo assembly are not well understood. Here we show that UNC-51/ATG1 kinase regulates the interaction between synaptic vesicles and motor complexes during transport in Drosophila. UNC-51 binds UNC-76, a kinesin heavy chain (KHC) adaptor protein. Loss of unc-51 or unc-76 leads to severe axonal transport defects in which synaptic vesicles are segregated from the motor complexes and accumulate along axons. Genetic studies show that unc-51 and unc-76 functionally interact in vivo to regulate axonal transport. UNC-51 phosphorylates UNC-76 on Ser(143), and the phosphorylated UNC-76 binds Synaptotagmin-1, a synaptic vesicle protein, suggesting that motor-cargo interactions are regulated in a phosphorylation-dependent manner. In addition, defective axonal transport in unc-76 mutants is rescued by a phospho-mimetic UNC-76, but not a phospho-defective UNC-76, demonstrating the essential role of UNC-76 Ser(143) phosphorylation in axonal transport. Thus, our data provide insight into axonal transport regulation that depends on the phosphorylation of adaptor proteins.

Spectroscopic approach for monitoring two-photon excited fluorescence resonance energy transfer from homodimers at the subcellular level.

We have employed a spectroscopic approach for monitoring fluorescence resonance energy transfer (FRET) in living cells. This method provides excellent spectral separation of green fluorescent protein (GFP) mutant signals within a subcellular imaging volume using two-photon excited fluorescence imaging and spectroscopy (TPIS-FRET). In contrast to current FRET-based methodologies, TPIS-FRET does not rely on the selection of optical filters, ratiometric image analysis, or bleedthrough correction algorithms. Utilizing the intrinsic optical sectioning capabilities of TPIS-FRET, we have identified protein-protein interactions within discrete subcellular domains. To illustrate the applicability of this technique to the detection of homodimer formation, we demonstrated the in vivo association of promyleocyte (PML) homodimers within their corresponding nuclear body.

Spatial Distribution and Function of Sterol Regulatory Element-Binding Protein 1a and 2 Homo- and Heterodimers by In Vivo Two-Photon Imaging and Spectroscopy Fluorescence Resonance Energy Transfer

ABSTRACT Sterol regulatory element-binding proteins (SREBPs) are a subfamily of basic helix-loop-helix-leucine zipper proteins that regulate lipid metabolism. We show novel evidence of the in vivo occurrence and subnuclear spatial localization of both exogenously expressed SREBP-1a and -2 homodimers and heterodimers obtained by two-photon imaging and spectroscopy fluorescence resonance energy transfer. SREBP-1a homodimers localize diffusely in the nucleus, whereas SREBP-2 homodimers and the SREBP-1a/SREBP-2 heterodimer localize predominantly to nuclear speckles or foci, with some cells showing a diffuse pattern. We also used tethered SREBP dimers to demonstrate that both homo- and heterodimeric SREBPs activate transcription in vivo. Ultrastructural analysis revealed that the punctate foci containing SREBP-2 are electron-dense nuclear bodies, similar or identical to structures containing the promyelocyte (PML) protein. Immunofluorescence studies suggest that a dynamic interplay exists between PML, as well as another component of the PML-containing nuclear body, SUMO-1, and SREBP-2 within these nuclear structures. These findings provide new insight into the overall process of transcriptional activation mediated by the SREBP family.

Association between promyelocyte protein and small ubiquitin-like modifier protein and the progression of cervical neoplasia.

OBJECTIVE To examine the association between the size and number of promyelocyte protein-containing nuclear bodies, their colocalization with the small ubiquitin-like modifier protein, and existing histopathologic staging of cervical neoplasia progressing toward squamous cell carcinoma. METHODS Fluorescence-based immunodetection of the promyelocyte protein and the small ubiquitin-like modifier protein was performed on paraffin-embedded and histopathologically graded human uterine cervical tissues. Quantitative measurements of the size and number of the promyelocyte protein-containing nuclear bodies were made and statistically analyzed. RESULTS We found that promyelocyte protein-containing nuclear bodies exhibit changes in both size and number throughout the continuum of cervical intraepithelial neoplasia (CIN) and cervical squamous cell carcinoma. An increase in number and size of the bodies occurs with progression from normal to CIN I/CIN II. In CIN III, two new subcategories of nuclear body are present with distinctly different promyelocyte protein patterns, with the type B CIN III losing the small ubiquitin-like modifier protein partnership. In squamous cell carcinoma, we see the loss of this colocalization in both well and poorly differentiated tumors, with a distinctly different promyelocyte protein pattern. Well-differentiated tumors have bigger nuclear bodies that are more numerous than those of the poorly differentiated tumors. CONCLUSION These data support the use of promyelocyte and small ubiquitin-like modifier proteins as a cytodiagnostic marker that parallels cervical cancer progression.

One-photon versus two-photon excited fluorescence resonance energy transfer of GFP fusion proteins

Understanding the function of a protein by following its dynamic interplay with other proteins in a living cell can contribute fundamentally to the overall cellular process or disease in which it participates. The principles of fluorescence resonance energy transfer serve as the basis for the development of new methodology which utilizes mutants of the green fluorescent protein (GFP). A major drawback in utilizing FRET as a means of determining protein interaction has been the overlap in spectra between the donor and acceptor GFP fluorophores and attempts to separate them by filters. To circumvent this issue, one-photon spectral data were generated for the FRET pairs expressed in living cells. To validate the protein-protein interaction we applied dequenching techniques whereby bleaching the acceptor fluorophore would lead to an increase or dequenching of the donor fluorescence. The FRET spectra were quantitatively compared as ratios of the donor and acceptor emission peaks (arbitrary intensities). In comparison, two-photon generated fluorescence of the FRET pairs provides for direct rationing of the intensity peaks, since at 810nm the donor is efficiently excited with the acceptor minimally excited. Furthermore, bleaching of the GFP molecules is negligible. Together, one-photon and two-photon excited FRET complimentarily provides proof of protein-protein interaction in living cells.

Utilization of two-photon FRET to monitor SREBP homodimer and heterodimer formation in living cells

Key players in cholesterol regulation are the members of a family of transcription factors known as the Sterol Regulatory Binding Proteins or SREBPs. The cellular redundancy of these proteins is under investigation, and our findings suggest that where these proteins reside may provide evidence for differences in the molecular dynamics of their transcriptional activity. Specifically, we have found that GFP-tagged SREBP-2 in contrast to SREBP-1 resides in discrete nuclear foci. To further explore functional differences between SREBP-1 and SREBP-2 we have developed an approach to monitor hetero- and homodimer formation by two-photon imaging and spectroscopy of fluorescence resonance energy transfer (TPIS-FRET). TPIS-FRET results will be presented. Collectively, these findings support the possibility that differences in function between SREBP family members may be governed by their localization within the cell.

Laser microbeam abalation of GFP-labeled nuclear organelles in a living cell

Cancer, development, cellular growth and differentiation are governed by gene expression. Recent molecular and cellular advances to visualize and perturb the pathways of transcriptional regulation, nascent RNA processing, and protein trafficking at the single cell level have been developed. More recently, applications utilizing the green fluorescent marker (GFP) from Aequorea victoria have facilitated visualization of these molecular events in a living cell. Specifically, we will describe a novel approach to perturb cellular processes by labeling discrete cellular components of interest with GFP and subsequently altering/ablating them with a laser microbeam.Cancer, development, cellular growth and differentiation are governed by gene expression. Recent molecular and cellular advances to visualize and perturb the pathways of transcriptional regulation, nascent RNA processing, and protein trafficking at the single cell level have been developed. More recently, applications utilizing the green fluorescent marker (GFP) from Aequorea victoria have facilitated visualization of these molecular events in a living cell. Specifically, we will describe a novel approach to perturb cellular processes by labeling discrete cellular components of interest with GFP and subsequently altering/ablating them with a laser microbeam.