Oligomeric viral proteins: small in size,large in presence

Viruses are obligate parasites that rely heavily on host cellular processes for replication. The small number of proteins typically encoded by a virus is faced with selection pressures that lead to the evolution of distinctive structural properties, allowing each protein to maintain its function under constraints such as small genome size, high mutation rate, and rapidly changing fitness conditions. One common strategy for this evolution is to utilize small building blocks to generate protein oligomers that assemble in multiple ways, thereby diversifying protein function and regulation. In this review, we discuss specific cases that illustrate how oligomerization is used to generate a single defined functional state, to modulate activity via different oligomeric states, or to generate multiple functional forms via different oligomeric states.     

Spatial Intensity Distribution Analysis Reveals Abnormal Oligomerization of Proteins in Single Cells

Knowledge of membrane receptor organization is essential for understanding the initial steps in cell signaling and trafficking mechanisms, but quantitative analysis of receptor interactions at the single-cell level and in different cellular compartments has remained highly challenging. To achieve this, we apply a quantitative image analysis technique—spatial intensity distribution analysis (SpIDA)—that can measure fluorescent particle concentrations and oligomerization states within different subcellular compartments in live cells. An important technical challenge faced by fluorescence microscopy-based measurement of oligomerization is the fidelity of receptor labeling. In practice, imperfect labeling biases the distribution of oligomeric states measured within an aggregated system. We extend SpIDA to enable analysis of high-order oligomers from fluorescence microscopy images, by including a probability weighted correction algorithm for nonemitting labels. We demonstrated that this fraction of nonemitting probes could be estimated in single cells using SpIDA measurements on model systems with known oligomerization state. Previously, this artifact was measured using single-step photobleaching. This approach was validated using computer-simulated data and the imperfect labeling was quantified in cells with ion channels of known oligomer subunit count. It was then applied to quantify the oligomerization states in different cell compartments of the proteolipid protein (PLP) expressed in COS-7 cells. Expression of a mutant PLP linked to impaired trafficking resulted in the detection of PLP tetramers that persist in the endoplasmic reticulum, while no difference was measured at the membrane between the distributions of wild-type and mutated PLPs. Our results demonstrate that SpIDA allows measurement of protein oligomerization in different compartments of intact cells, even when fractional mislabeling occurs as well as photobleaching during the imaging process, and reveals insights into the mechanism underlying impaired trafficking of PLP.     

The cysteines of the extracellular loop are crucial for trafficking of human organic cation transporter 2 to the plasma membrane and are involved in oligomerization

Human organic cation transporter 2 (hOCT2) is involved in transport of many endogenous and exogenous organic cations, mainly in kidney and brain cells. Because the quaternary structure of transmembrane proteins plays an essential role for their cellular trafficking and function, we investigated whether hOCT2 forms oligomeric complexes, and if so, which part of the transporter is involved in the oligomerization. A yeast 2-hybrid mating-based split-ubiquitin system (mbSUS), fluorescence resonance energy transfer, Western blot analysis, cross-linking experiments, immunofluorescence, and uptake measurements of the fluorescent organic cation 4-(4-(dimethylamino)styryl)-N-methylpyridinium were applied to human embryonic kidney 293 (HEK293) cells transfected with hOCT2 and partly also to freshly isolated human proximal tubules. The role of cysteines for oligomerization and trafficking of the transporter to the plasma membranes was investigated in cysteine mutants of hOCT2. hOCT2 formed oligomers both in the HEK293 expression system and in native human kidneys. The cysteines of the large extracellular loop are important to enable correct folding, oligomeric assembly, and plasma membrane insertion of hOCT2. Mutation of the first and the last cysteines of the loop at positions 51 and 143 abolished oligomer formation. Thus, the cysteines of the extracellular loop are important for correct trafficking of the transporter to the plasma membrane and for its oligomerization.     

Lectin-like oxidized low-density lipoprotein receptor (LOX-1) functions as an oligomer and oligomerization is dependent on receptor density

Lectin-like oxidized low-density lipoprotein (LDL) receptor (LOX-1) exists as a homodimer formed by an intermolecular disulfide bond. Although the dimer is the minimum structural unit of LOX-1 on cell membranes, LOX-1 can form larger noncovalent oligomeric complexes. But, the functional unit of LOX-1 is not known. We quantitatively analyzed the correlation between cyan fluorescent protein-tagged LOX-1 expression and the fluorescence-labeled ligand (DiD-AcLDL) binding ability on each cell. The results clearly indicate that there is a threshold level of expression that enables LOX-1 to bind ligand. Above this threshold level, the ability of LOX-1 to bind ligand was proportional to its level of expression. Using the membrane impermeable crosslinker BS(3), we detected oligomers (primarily hexamers) only on the cell lines that stably expressed LOX-1 above the threshold level. In contrast, little oligomer or ligand binding was detected in cell lines expressing LOX-1 below the threshold level. Moreover, oligomerization was independent of ligand binding. These results indicate that the functional unit of LOX-1 is an oligomer and that oligomerization of LOX-1 is dependent on the receptor density on the plasma membrane.     

The potassium channel KcsA: a model protein in studying membrane protein oligomerization and stability of oligomeric assembly?

Many membrane proteins are functional as stable oligomers. An understanding of the conditions that elicit and enhance oligomerization is important in many therapeutics. In this regard, protein–protein and protein–lipid interactions play crucial roles in the assembly and stability of oligomeric complexes. Recent years have seen a rapid increase in the mechanistic information on the importance of cytoplasmic termini in determining subunit assembly and stability of oligomeric complexes. In addition, the role of specific protein–lipid interaction between anionic phospholipids and “hot spots” on the protein surface has also become evident in stabilizing oligomeric assemblies. This review focuses on several contemporary developments of membrane proteins that stabilize oligomers by taking the potassium channel KcsA as an exemplary ion channel.     

Determination of membrane protein transporter oligomerization in native tissue using spatial fluorescence intensity fluctuation analysis

Membrane transporter proteins exist in a complex dynamic equilibrium between various oligomeric states that include monomers, dimers, dimer of dimers and higher order oligomers. Given their sub-optical microscopic resolution size, the oligomerization state of membrane transporters is difficult to quantify without requiring tissue disruption and indirect biochemical methods. Here we present the application of a fluorescence measurement technique which combines fluorescence image moment analysis and spatial intensity distribution analysis (SpIDA) to determine the oligomerization state of membrane proteins in situ. As a model system we analyzed the oligomeric state(s) of the electrogenic sodium bicarbonate cotransporter NBCe1-A in cultured cells and in rat kidney. The approaches that we describe offer for the first time the ability to investigate the oligomeric state of membrane transporter proteins in their native state.     

The Human Acid-Sensing Ion Channel ASIC1a: Evidence for a Homotetrameric Assembly State at the Cell Surface

The chicken acid-sensing ion channel ASIC1 has been crystallized as a homotrimer. We address here the oligomeric state of the functional ASIC1 in situ at the cell surface. The oligomeric states of functional ASIC1a and mutants with additional cysteines introduced in the extracellular pore vestibule were resolved on SDS-PAGE. The functional ASIC1 complexes were stabilized at the cell surface of Xenopus laevis oocytes or CHO cells either using the sulfhydryl crosslinker BMOE, or sodium tetrathionate (NaTT). Under these different crosslinking conditions ASIC1a migrates as four distinct oligomeric states that correspond by mass to multiples of a single ASIC1a subunit. The relative importance of each of the four ASIC1a oligomers was critically dependent on the availability of cysteines in the transmembrane domain for crosslinking, consistent with the presence of ASIC1a homo-oligomers. The expression of ASIC1a monomers, trimeric or tetrameric concatemeric cDNA constructs resulted in functional channels. The resulting ASIC1a complexes are resolved as a predominant tetramer over the other oligomeric forms, after stabilization with BMOE or NaTT and SDS-PAGE/western blot analysis. Our data identify a major ASIC1a homotetramer at the surface membrane of the cell expressing functional ASIC1a channel.     

Detyrosinated Glu-tubulin is a substrate for cellular Factor XIIIA transglutaminase in differentiating osteoblasts

Microtubule components α- and β-tubulin undergo a number of posttranslational modifications that modulate their dynamics and cellular functions. These modifications include polyamination and covalent crosslinking by transglutaminase enzymes. We have demonstrated previously that the less dynamic and more stable tubulin form—detyrosinated Glu-tubulin—is found in high molecular weight, oligomeric complexes in bone-forming osteoblasts during differentiation and along with deposition of collagenous extracellular matrix. In this study, we report that oligomeric Glu-tubulin has high nocodazole tolerance, indicating further increased stability. We show that α-tubulin, which gives rise to Glu-tubulin, is a transglutaminase substrate in in vitro assays and that it is crosslinked into oligomers (dimers, trimers and tetramers) by transglutaminase 2 and Factor XIIIA; β-tubulin was not crosslinked by transglutaminase activity. The oligomeric Glu-tubulin was specifically localized to the plasma membrane of osteoblasts as analyzed by subcellular fractionation, cell surface biotinylation experiments and total internal reflection fluorescence (TIRF) microscopy. Glu- and α-tubulin co-localized with cellular Factor XIIIA as analyzed by conventional and TIRF microscopy. The Factor XIIIA-specific substrate peptide bF11 co-localized with α-tubulin and acted as a competitive inhibitor to oligomerization of Glu-tubulin, attenuating its formation in cells. This was associated with significantly decreased type I collagen deposition and decreased secretory activity as measured by synaptotagmin VII levels on the osteoblast plasma membrane. Our results suggest that Glu-tubulin may exist as covalently stabilized form which may be linked to the secretion and elaboration of collagenous extracellular matrix.     

Investigation of ebola VP40 assembly and oligomerization in live cells using number and brightness analysis

Ebola virus assembles and buds from the inner leaflet of the plasma membrane of mammalian cells, which is primarily attributed to its major matrix protein VP40. Oligomerization of VP40 has been shown to be essential to the life cycle of the virus including formation of virions from infected cells. To date, VP40 oligomerization has mainly been assessed by chemical cross-linking following cell fractionation studies with VP40 transfected cells. This has made it difficult to discern the spatial and temporal dynamics of VP40 oligomerization. To gain a better understanding of the VP40 assembly and oligomerization process in live cells, we have employed real-time imaging of enhanced green fluorescent protein tagged VP40. Here, we use both confocal and total internal reflection microscopy coupled with number and brightness analysis to show that VP40 oligomers are localized on the plasma membrane and are highly enriched at sites of membrane protrusion, consistent with sites of viral budding. These filamentous plasma membrane protrusion sites harbor VP40 hexamers, octamers, and higher order oligomers. Consistent with previous reports, abrogation of VP40 oligomerization through mutagenesis greatly diminished VP40 egress and also abolished membrane protrusion sites enriched with VP40. In sum, real-time single-molecule imaging of fluorescently labeled Ebola VP40 is able to resolve the spatial and temporal dynamics of VP40 oligomerization.     

Raft-targeting and oligomerization of Parasporin-2, a bacillus thuringiensis crystal protein with anti-tumour activity

Parasporin-2 is a newly classified Bacillus thuringiensis crystal toxin with strong cytocidal activities toward human liver and colon cancer cells. Similar to other insecticidal B. thuringiensis crystal toxins, parasporin-2 shows target specificity and damages the cellular membrane. However, the mode of parasporin-2 actions toward the cell membrane remains unknown. Here, we show that this anti-tumour crystal toxin targets lipid rafts and assembles into oligomeric complexes in the membrane of human hepatocyte cancer (HepG2) cells. Upon incubation with HepG2 cells, peripheral membrane-bound toxins, which were recovered in a low-density detergent-resistant membrane fraction, i.e. with lipid rafts, were transformed into heat-stable SDS-resistant membrane-embedded oligomers (approximately 200 kDa). The toxin oligomerization was dependent on temperature and coupled with cell lysis. The toxin oligomerization also occurred in a cell-free membrane system and was required for binding to membrane proteins, the lipid bilayer and cholesterols. These results indicate that parasporin-2 is an oligomerizing and pore-forming toxin that accumulates in lipid rafts.     

Efficient coupling of transducin to monomeric rhodopsin in a phospholipid bilayer

G protein-coupled receptors (GPCRs) are seven transmembrane domain proteins that transduce extracellular signals across the plasma membrane and couple to the heterotrimeric family of G proteins. Like most intrinsic membrane proteins, GPCRs are capable of oligomerization, the function of which has only been established for a few different receptor systems. One challenge in understanding the function of oligomers relates to the inability to separate monomeric and oligomeric receptor complexes in membrane environments. Here we report the reconstitution of bovine rhodopsin, a GPCR expressed in the retina, into an apolipoprotein A-I phospholipid particle, derived from high density lipoprotein (HDL). We demonstrate that rhodopsin, when incorporated into these 10 nm reconstituted HDL (rHDL) particles, is monomeric and functional. Rhodopsin.rHDL maintains the appropriate spectral properties with respect to photoactivation and formation of the active form, metarhodopsin II. Additionally, the kinetics of metarhodopsin II decay is similar between rhodopsin in native membranes and rhodopsin in rHDL particles. Photoactivation of monomeric rhodopsin.rHDL also results in the rapid activation of transducin, at a rate that is comparable with that found in native rod outer segments and 20-fold faster than rhodopsin in detergent micelles. These data suggest that monomeric rhodopsin is the minimal functional unit in G protein activation and that oligomerization is not absolutely required for this process.     

Membrane assembly of simple helix homo-oligomers studied via molecular dynamics simulations

The assembly of simple transmembrane helix homo-oligomers is studied by combining a generalized Born implicit membrane model with replica exchange molecular dynamics simulations to sample the conformational space of various oligomerization states and the native oligomeric conformation. Our approach is applied to predict the structures of transmembrane helices of three proteins--glycophorin A, the M2 proton channel, and phospholamban--using only peptide sequence and the native oligomerization state information. In every case, the methodology reproduces native conformations that are in good agreement with available experimental structural data. Thus, our method should be useful in the prediction of native structures of transmembrane domains of other peptides. When we ignore the experimental constraint on the native oligomerization state and attempt de novo prediction of the structure and oligomerization state based only on sequence and simple energetic considerations, we identify the pentamer as the most stable oligomer for phospholamban. However, for the glycophorin A and the M2 proton channels, we tend to predict higher oligomers as more stable. Our studies demonstrate that reliable predictions of the structure of transmembrane helical oligomers can be achieved when the observed oligomerization state is imposed as a constraint, but that further efforts are needed for the de novo prediction of both structure and oligomeric state.     

Single molecule imaging of green fluorescent proteins in living cells: E-cadherin forms oligomers on the free cell surface

Single green fluorescent protein (GFP) molecules were successfully imaged for the first time in living cells. GFP linked to the cytoplasmic carboxyl terminus of E-cadherin (E-cad-GFP) was expressed in mouse fibroblast L cells, and observed using an objective-type total internal reflection fluorescence microscope. Based on the fluorescence intensity of individual fluorescent spots, the majority of E-cad-GFP molecules on the free cell surface were found to be oligomers of various sizes, many of them greater than dimers, suggesting that oligomerization of E-cadherin takes place before its assembly at cell-cell adhesion sites. The translational diffusion coefficient of E-cad-GFP is reduced by a factor of 10 to 40 upon oligomerization. Because such large decreases in translational mobility cannot be explained solely by increases in radius upon oligomerization, an oligomerization-induced trapping model is proposed in which, when oligomers are formed, they are trapped in place due to greatly enhanced tethering and corralling effects of the membrane skeleton on oligomers (compared with monomers). The presence of many oligomers greater than dimers on the free surface suggests that these greater oligomers are the basic building blocks for the two-dimensional cell adhesion structures (adherens junctions).     

Caught in self-interaction: evolutionary and functional mechanisms of protein homooligomerization

Many soluble and membrane proteins form homooligomeric complexes in a cell which are responsible for the diversity and specificity of many pathways, may mediate and regulate gene expression, activity of enzymes, ion channels, receptors, and cell adhesion processes. The evolutionary and physical mechanisms of oligomerization are very diverse and its general principles have not yet been formulated. Homooligomeric states may be conserved within certain protein subfamilies and might be important in providing specificity to certain substrates while minimizing interactions with other unwanted partners. Moreover, recent studies have led to a greater awareness that transitions between different oligomeric states may regulate protein activity and provide the switch between different pathways. In this paper we summarize the biological importance of homooligomeric assemblies, physico-chemical properties of their interfaces, experimental and computational methods for their identification and prediction. We particularly focus on homooligomer evolution and describe the mechanisms to develop new specificities through the formation of different homooligomeric complexes. Finally, we discuss the possible role of oligomeric transitions in the regulation of protein activity and compile a set of experimental examples with such regulatory mechanisms.     

Ligand-dependent inhibition of oligomerization at the human thyrotropin receptor

Recently, several studies have reported oligomerization of G protein-coupled receptors, although the functional implications of this phenomenon are still unclear. Using fluorescence resonance energy transfer (FRET) and coimmunoprecipitation (COIP), we previously reported that the human thyrotropin (TSH) receptor tagged with green fluorescent protein (TSHR(GFP)) and expressed in a heterologous system was present as oligomeric complexes on the cell surface. Here, we have extended this biophysical and biochemical approach to study the regulation of such oligomeric complexes. Co-expression of TSHR(GFP) and TSHR(Myc) constructs in Chinese hamster ovary cells resulted in FRET-positive cells. The specificity of the FRET signal was verified by the absence of energy transfer in individually transfected TSHR(GFP) and TSHR(Myc):Cy3 cells cultured together and also by acceptor photobleaching. Occupation of the receptor molecule by the ligand (TSH) resulted in a dose-dependent decrease in the FRET index from 20% in the absence of TSH to <1% with 10(3) microunits/ml of TSH. Such reduction in oligomeric forms was also confirmed by coimmunoprecipitation. Exposure of TSHR(GFP/Myc) cells to forskolin or cytochalasin D caused no change in the FRET index, confirming that the decrease in the oligomeric complexes was a receptor-dependent phenomenon and free of energy or microtuble requirements. The TSH-induced decrease in TSHR oligomers was found to be secondary to dissociation of the TSHR complexes as evidenced by an increase in fluorescent intensity of photobleached spots of GFP fluorescence with 10(3) microunits/ml of TSH. These data indicated that the less active conformation of the TSHR was comprised of receptor complexes and that such complexes were dissociated on the binding of ligand. Such observations support the concept of a constitutively active TSHR dimer or monomer that is naturally inhibited by the formation of higher order complexes. Inhibition of these oligomeric forms by ligand binding returns the TSHR to an activated state.     

Oligomerization of dopamine transporters visualized in living cells by fluorescence resonance energy transfer microscopy

To examine the oligomeric state and trafficking of the dopamine transporter (DAT) in different compartments of living cells, human DAT was fused to yellow (YFP) or cyan fluorescent protein (CFP). YFP-DAT and CFP-DAT were transiently and stably expressed in porcine aortic endothelial (PAE) cells, human embryonic kidney (HEK) 293 cells, and an immortalized dopaminergic cell line 1RB3AN27. Fluorescence microscopic imaging of cells co-expressing YFP-DAT and CFP-DAT revealed fluorescence resonance energy transfer (FRET) between CFP and YFP, which is consistent with an intermolecular interaction of DAT fusion proteins. FRET signals were detected between CFP- and YFP-DAT located at the plasma membrane and in intracellular membrane compartments. Phorbol esters or amphetamine induced the endocytosis of YFP/CFP-DAT to early and recycling endosomes, identified by Rab5, Rab11, Hrs and EEA.1 proteins. Interestingly, however, DAT was mainly excluded from Rab5- and Hrs-containing microdomains within the endosomes. The strongest FRET signals were measured in endosomes, indicative of efficient oligomerization of internalized DAT. The intermolecular DAT interactions were confirmed by co-immunoprecipitation. A DAT mutant that was retained in the endoplasmic reticulum (ER) after biosynthesis was used to show that DAT is oligomeric in the ER. Moreover, co-expression of an ER-retained DAT mutant and wild-type DAT resulted in the retention of wild-type DAT in the ER. These data suggest that DAT oligomers are formed in the ER and then are constitutively maintained both at the cell surface and during trafficking between the plasma membrane and endosomes.     

Antimicrobial activity profiles of Amphiphilic Xanthone derivatives are a function of their molecular Oligomerization

Currently, membrane-targeting small antimicrobial peptidomimetics (SAP) are important in antibiotic development because bacteria appear to develop resistance to these surface-active compounds less readily. However, the molecular membrane-targeting action of SAPs has received little attention. In this study, we investigated the effect of oligomerization of amphiphilic xanthone, a model SAP, on its antimicrobial properties against both Gram-positive and Gram-negative bacteria. First, oligomer formation by an amphiphilic xanthone, compound 2 (also coded as AM052), was investigated via solution-state nuclear magnetic resonance (NMR) spectroscopy. Then, the effects of oligomerization on membrane disruption were further studied via biophysical approaches. The results showed that the antimicrobial activities of SAPs develop in several stages: oligomer formation in aqueous solution, initial binding of oligomers to the membrane-water interface followed by insertion into the membrane bilayer, aggregation of antimicrobial oligomers in the membrane, and induced membrane leakage. Ultimately, the presence of the oligomers in the bacterial membrane leads to decreased membrane fluidity and bacterial cell death. Interestingly, the early formation of large oligomers leads to stronger membrane disruption and more rapid bacterial killing. However, reduced antimicrobial activities against Gram-negative bacteria were observed for compounds that formed larger oligomers because the LPS layer acts as a barrier to large complexes. Taken together, our results suggest that oligomerization of SAPs has a strong impact on their antimicrobial properties.     

Quantifying Protein-Protein Interactions of Peripheral Membrane Proteins by Fluorescence Brightness Analysis

Fluorescently labeled proteins that are found both in the cytoplasm and at the plasma membrane, such as peripheral membrane proteins, create stratified fluorescent layers that present a challenging environment for brightness studies with fluorescence fluctuation spectroscopy. The geometry of each layer along with fluorescence and brightness contributions from adjacent layers generates a convoluted raw brightness that conceals the underlying brightness of each individual layer. Because the brightness at a layer establishes the oligomeric state of the fluorescently labeled protein at said layer, we developed a method that connects the experimental raw brightness with the physical brightness at each layered compartment. The technique determines the oligomerization in each compartment from an axial intensity scan through the sample, followed by a fluorescence fluctuation spectroscopy measurement at each layer. We experimentally verify the technique with H-Ras-EGFP as a model system and determine its oligomeric state at both the plasma membrane and in the cytoplasm. Furthermore, we study the oligomerization of the Gag matrix domain of Human T-lymphotropic virus Type 1. The matrix domain targets the Gag polyprotein to the plasma membrane where, subsequently, viral assembly occurs. We determine the oligomerization of matrix in the cytoplasm and observe the onset of protein-protein interactions at the membrane. These observations shed light on the early assembly steps of the retrovirus.     

Glycine residues G338 and G342 are important determinants for serotonin transporter dimerisation and cell surface expression

Compelling evidence has been provided that Na(+) and Cl(-)-dependent neurotransmitter transporter proteins form oligomeric complexes. Specific helix-helix interactions in lipid bilayers are thought to promote the assembly of integral membrane proteins to oligomeric structures. These interactions are determined by selective transmembrane helix packing motifs one of which is the Glycophorin A motif (GxxxG). This motif is present in the sixth transmembrane domain of most transporter proteins. In order to investigate, whether this motif is important for proper expression and function of the serotonin transporter (SERT), we have analysed the effect of mutating the respective glycine residues Gly338 and Gly342 to valine upon transient expression of the respective cDNAs in HEK293 cells. As revealed by western blotting, wildtype SERT is found in monomeric and dimeric forms while both mutants are expressed as monomers solely. Confocal microscopy revealed that the wildtype SERT is expressed at the cell surface, whereas both mutant proteins are localised in intracellular compartments. Failure of integration into the cell membrane is responsible for a total loss of [(3)H]5HT uptake capability by the mutants. These findings show that in the SERT protein the integrity of the GxxxG motif is essential for dimerisation and proper targeting of the transporter complex to the cell surface.     

Quantifying Protein-Protein Interactions of Peripheral Membrane Proteins by Fluorescence Brightness Analysis

Fluorescently labeled proteins that are found both in the cytoplasm and at the plasma membrane, such as peripheral membrane proteins, create stratified fluorescent layers that present a challenging environment for brightness studies with fluorescence fluctuation spectroscopy. The geometry of each layer along with fluorescence and brightness contributions from adjacent layers generates a convoluted raw brightness that conceals the underlying brightness of each individual layer. Because the brightness at a layer establishes the oligomeric state of the fluorescently labeled protein at said layer, we developed a method that connects the experimental raw brightness with the physical brightness at each layered compartment. The technique determines the oligomerization in each compartment from an axial intensity scan through the sample, followed by a fluorescence fluctuation spectroscopy measurement at each layer. We experimentally verify the technique with H-Ras-EGFP as a model system and determine its oligomeric state at both the plasma membrane and in the cytoplasm. Furthermore, we study the oligomerization of the Gag matrix domain of Human T-lymphotropic virus Type 1. The matrix domain targets the Gag polyprotein to the plasma membrane where, subsequently, viral assembly occurs. We determine the oligomerization of matrix in the cytoplasm and observe the onset of protein-protein interactions at the membrane. These observations shed light on the early assembly steps of the retrovirus.