Presence Of Membrane Proteins Research Articles

Polymyxin B1 in the Escherichia coli inner membrane: A complex story of protein and lipopolysaccharide-mediated insertion

The rise in multi-drug resistant Gram-negative bacterial infections has led to an increased need for 'last-resort' antibiotics such as polymyxins. However, the emergence of polymyxin-resistant strains threatens to bring about a post-antibiotic era. Thus, there is a need to develop new polymyxin-based antibiotics, but a lack of knowledge of the mechanism of action of polymyxins hinders such efforts. It has recently been suggested that polymyxins induce cell lysis of the Gram-negative bacterial inner membrane (IM) by targeting trace amounts of lipopolysaccharide (LPS) localized there. We use multiscale molecular dynamics (MD) including long-timescale coarse-grained (CG) and all-atom (AA) simulations to investigate the interactions of polymyxin B1 (PMB1) with bacterial IM models containing phospholipids (PLs), small quantities of LPS, and IM proteins. LPS was observed to (transiently) phase separate from PLs at multiple LPS concentrations, and associate with proteins in the IM. PMB1 spontaneously inserted into the IM and localized at the LPS-PL interface, where it cross-linked lipid headgroups via hydrogen bonds, sampling a wide range of interfacial environments. In the presence of membrane proteins, a small number of PMB1 molecules formed interactions with them, in a manner that was modulated by local LPS molecules. Electroporation-driven translocation of PMB1 via water-filled pores was favored at the protein-PL interface, supporting the 'destabilizing' role proteins may have within the IM. Overall, this in-depth characterization of PMB1 modes of interaction reveals how small amounts of mislocalized LPS may play a role in pre-lytic targeting and provides insights that may facilitate rational improvement of polymyxin-based antibiotics.

Limited miscibility in hydrated DPPC – Lyso-PPC systems

Hydrated dipalmitoylphosphatidylcholine (DPPC) and lyso-palmitoylphosphatidylcholine (Lyso-PPC, abbreviated as LPC) mixtures form vesicles composed from periodically or randomly arranged layers and micelle-like nanoparticles, as revealed by a wide range of thermodynamical, structural, and morphological characterization methods. Based on five different experimental methods, we have reconstructed the phase diagram of the system. The micelle-like objects are strongly anisotropic and are identified as bicelles. Even a small amount of LPC induces drastic changes in the thermotropic phase behaviour of DPPC. Here, an interdigitated gel (LβI) structure, coexisting with the other gel phases, forms at moderate LPC concentration. Bicelles and LβI structured vesicles appear in the equimolar LPC ratio range (0.4 ≤ XLPC ≤ 0.6). Two complex phase transition regimes are detectable. Between 36–40 °C, bicelles are pre-melted while domains of LβI structure are “crystallized” through local endothermic and exothermic transitions, respectively. Then, around 42 °C the chains of both the LβI and bicelles melt completely. Above the equimolar LPC ratio range (XLPC > 0.6) the chain melting in the LβI structure vanishes, while this transition still exists in the chain region of bicelles. This wealth of structural forms, due to the weak first order transition characteristics, is highly sensitive for perturbations and interactions induced by e.g. the presence of membrane proteins.

Expression, purification and micelle reconstruction of the transmembrane domain of the human amyloid precursor protein for NMR studies

The multiple-step cleavage of amyloid precursor protein (APP) generates amyloid-β peptides (Aβ), highly toxic molecules causing Alzheimer's disease (AD). The nonspecific cleavage between the transmembrane region of APP (APPTM) and γ-secretase is the key step of Aβ generation. Reconstituting APPTM under physiologically-relevant conditions is crucial to investigate how it interacts with γ-secretase and for future AD drug discovery. Although producing recombinant APPTM was reported before, the large scale purification was hindered by the use of biological protease in the presence of membrane protein. Here, we expressed recombinant APPTM in Escherichia coli using the pMM-LR6 vector and recovered the fusion protein from inclusion bodies. By combining Ni-NTA chromatography, cyanogen bromide cleavage, and reverse phase high performance liquid chromatography (RP-HPLC), isotopically-labeled APPTM was obtained in high yield and high purity. The reconstitution of APPTM into dodecylphosphocholine (DPC) micelle generated mono dispersed 2D 15N-1H HSQC spectra in high quality. We successfully established an efficient and reliable method for the expression, purification and reconstruction of APPTM, which may facilitate future investigation of APPTM and its complex in more native like membrane mimetics such as bicelle and nanodiscs.

Interactions of polymyxin B1 with the gram-negative inner membrane: A simulation study.

The rise of drug resistant Gram-negative bacterial infections in recent decades has led to increased use of polymyxins, a class of antimicrobial peptides, as a ‘last resort’ treatment. In addition, the recent emergence of polymyxin-resistant strains threatens to bring about an era in which bacteria are resistant to all known antibiotics. It is thus necessary to develop new polymyxin-based antibiotics, but a lack of understanding of how polymyxins work at the molecular level may hinder such efforts. One question that remains open is how do polymyxins interact with the Gram-negative inner membrane to cause cell lysis? It has recently been suggested that polymyxins may target small quantities of the complex glycolipid lipopolysaccharide (LPS) that are present in the inner membrane prior to LPS transport to the outer membrane. In this work, we used a multiscale molecular dynamics (MD) approach including long-timescale, coarse-grained (CG) and atomistic simulations to investigate the interactions of polymyxin B1 (PMB1) with bacterial inner membrane models in the presence of small quantities of LPS. We observed that PMB1 is inserted into the inner membrane and localises at the LPS-phospholipid interface, sampling a wide range of interfacial environments. Our CG simulations also indicate that PMB1-LPS interactions are long-lived and numerous. In the presence of membrane proteins, a small number of PMB1 molecules were observed to form interactions with native inner membrane proteins, reducing the overall probability of PMB1 molecules remaining bound to LPS. Overall, this work indicates that PMB1 can target LPS molecules in the inner membrane, supporting recent hypotheses on its mode of action, while also providing new insights into the effect of membrane proteins upon PMB1-inner membrane interactions.

Symposium review: The relevance of bovine milk phospholipids in human nutrition—Evidence of the effect on infant gut and brain development

This paper reflects the concepts reviewed during the presentation in the Joint MILK/Lactation Biology Symposium at the ADSA 2018 Annual Meeting. Our intention is to update the concepts and advances in the area of research regarding milk phospholipids or polar lipid fraction as part of a dairy ingredient used today in nutritional studies that focus on gut health as well as brain development of infants. Although processing advances have allowed the production of novel ingredients rich in milk fat globule membrane (MFGM) components, mostly monitored by phospholipid concentration and presence of membrane proteins, there is wide variability in their composition and structure. Furthermore, we aimed to include in the phospholipid fraction of milk nanovesicles designated as milk exosomes, which are secreted into milk by different secretion mechanisms than those of the fat globules but are also made up of a unique mixture of polar lipids. We consider imperative the study of polar lipid-derived structures from milk regarding composition and structure to gain insights into their biological effect in human health. Nevertheless, and tolerating the differences in composition and concentration of their components, studies supplementing the diet of infants with polar lipids (i.e., MFGM components) have shown significant advances in several areas of human health and well-being. Here we present a summary of the important components of MFGM and milk exosomes as well as an overview of the effects on gut health and brain and cognitive development when added to the diet of infants.

Investigation of the morphological transition of a phospholipid bilayer membrane in an external electric field via molecular dynamics simulation.

Elucidating the mechanisms for morphological transitions of the phospholipid bilayer membrane during cellular activity should lead to greater understanding of these membrane transitions and allow us to optimize biotechnologies such as drug delivery systems in organisms. To investigate the mechanism for and the dynamics of morphological changes in the phospholipid membrane, we performed molecular dynamics simulation of a phospholipid membrane with and without membrane protein under the influence of electric fields with different strengths. In the absence of membrane protein, it was possible to control the transition from one lamellar membrane morphology to another by applying a strong electric field. The strong electric field initially disordered the lipid molecules in the membrane, leading to the formation of a hydrophilic pore. The lipid molecules then spontaneously fused into a new lamellar membrane morphology. In the presence of membrane protein, a morphological transition from lamellar membrane to vesicle under the influence of a strong electric field was observed. Studying the complex transition dynamics associated with these changes in membrane morphology allowed us to gain deep insight into the electrofusion and electroporation that occur in the presence or absence of membrane protein, and the results obtained here should prove useful in work aimed at controlling membrane morphology. Graphical Abstract Memebrane morphological transition under the electric field of 0.6 V/nm with the membrane protein (down) and without membrane protein (up).

Extension of the Highly Mobile Membrane Mimetic to Transmembrane Systems through Customized in Silico Solvents.

The mechanics of the protein-lipid interactions of transmembrane proteins are difficult to capture with conventional atomic molecular dynamics, due to the slow lateral diffusion of lipids restricting sampling to states near the initial membrane configuration. The highly mobile membrane mimetic (HMMM) model accelerates lipid dynamics by modeling the acyl tails nearest to the membrane center as a fluid organic solvent while maintaining an atomic description of the lipid headgroups and short acyl tails. The HMMM has been applied to many peripheral protein systems; however, the organic solvent used to date caused deformations in transmembrane proteins by intercalating into the protein and disrupting interactions between individual side chains. We ameliorate the effect of the solvent on transmembrane protein structure through the development of two new in silico Lennard-Jones solvents. The parameters for the new solvents were determined through an extensive parameter search in order to match the bulk properties of alkanes in a highly simplified model. Using these new solvents, we substantially improve the insertion free energy profiles of 10 protein side chain analogues across the entire bilayer. In addition, we reduce the intercalation of solvent into transmembrane systems, resulting in native-like transmembrane protein structures from five different topological classes within a HMMM bilayer. The parametrization of the solvents, in addition to their computed physical properties, is discussed. By combining high lipid lateral diffusion with intact transmembrane proteins, we foresee the developed solvents being useful to efficiently identify membrane composition inhomogeneities and lipid binding caused by the presence of membrane proteins.

Membrane mechanical properties of synthetic asymmetric phospholipid vesicles.

Synthetic lipid vesicles have served as important model systems to study cellular membrane biology. Research has shown that the mechanical properties of bilayer membranes significantly affects their biological behavior. The properties of a lipid bilayer are governed by lipid acyl chain length, headgroup type, and the presence of membrane proteins. However, few studies have explored how membrane architecture, in particular trans-bilayer lipid asymmetry, influences membrane mechanical properties. In this study, we investigated the effects of lipid bilayer architecture (i.e. asymmetry) on the mechanical properties of biological membranes. This was achieved using a customized micropipette aspiration system and a novel microfluidic technique previously developed by our team for building asymmetric phospholipid vesicles with tailored bilayer architecture. We found that the bending modulus and area expansion modulus of the synthetic asymmetric bilayers were up to 50% larger than the values acquired for symmetric bilayers. This was caused by the dissimilar lipid distribution in each leaflet of the bilayer for the asymmetric membrane. To the best of our knowledge, this is the first report on the impact of trans-bilayer asymmetry on the area expansion modulus of synthetic bilayer membranes. Since the mechanical properties of bilayer membranes play an important role in numerous cellular processes, these results have significant implications for membrane biology studies.

Aggregation, fusion, and leakage of liposomes induced by peptides.

Biological membranes are heterogeneous systems. Their functions are closely related to the lipid lateral segregation in the presence of membrane proteins. In this work, we designed two peptides, amphiphilic cationic peptides K3L8K3 and nonamphiphilic peptides K20, and studied their interactions with binary liposomes in different phases (Lα, Lβ', and Lα/Lβ'). As mimics of membrane proteins, both K3L8K3 and K20 can cause the liposomes to aggregate, fuse, or leak. These processes were closely related to the phases of liposomes. For the liposomes in Lα phase, heavy aggregation, fusion, and leakage were observed in the presence of either K20 or K3L8K3. For the liposomes in Lβ' phase, neither K3L8K3 nor K20 can induce fusion or leakage. For the liposomes in Lα/Lβ' phase, K3L8K3 caused the liposomes to aggregate, fuse, and leak, while K20 only led to aggregation. The kinetics of aggregation, fusion, and leakage in each phase were recorded, and they were related to the lipid demixing in the presence of the peptide. Our work not only gained insight into the effect of the lipid demixing on the interactions between peptide and membrane, but also helped in developing drug delivery vehicles with liposomes as the platform.

Development and characterization of a multi‐drug resistant Her‐2/neu positive breast cancer cell line (58.6)

Research has shown that ~ 25% ‐ 30% breast cancers overexpress Her‐2/neu receptor. The major problem in the management of Her‐2/neu overexpressed breast cancer is its multi‐drug resistantce (MDR). P‐glycoprotein (P‐gp) mediated MDR has been recognized as one of the major mechanisms in drug resistance in breast cancer. Therefore, developing a breast cancer cell line with both Her‐2/neu and MDR positive becomes necessary.Dual positive breast cancer cell line was developed by transfection of the multidrug resistance gene (MDR1 gene) to human breast carcinoma cell line BT‐474 (Her‐2/neu +), followed by selection on clochicine. The presence of membrane protein Her‐2 and P‐gp were tested by immunohistochemical methods. The multidrug resistance was tested by the treatment efficacy of doxorubicin.The overexpression of p‐glycoprotein in multidrug resistant cells and the presence of membrane protein Her‐2 were confirmed by immunocytochemistry staining as well as Western Blot. Fluorescence imaging study showed that drug resistant BT‐474 cells uptake much more Her‐2/neu tagged nanoparticles than non‐tagged ones. Cytotoxicity test shows that the transfected BT‐474 is 10 times more resistant than its drug sensitive counterpart.In conclusion, we have successfully developed and characterized Her‐2/neu and MDR dual positive breast cancer cell line which can serve as a platform for testing new therapeutic strategies.Grant Funding Source: Supported by Susan G. Komen for the Cure foundation

2H Solid-State NMR Studies of the Antimicrobial Peptide MSI-78 Interacting with the Membranes of Whole Escherichia Coli

Antimicrobial peptides (AMPs) are ubiquitous molecules that can display antimicrobial activity against bacteria, viruses, protozoa and various other pathogens. A key aspect of AMP activity is their interactions with biological membranes. Solid-state NMR spectroscopy has thus been an important tool for their study. Previous experiments, on model membrane systems, have elucidated important aspects of AMP mechanism. However, the extent to which actual in vivo AMP activity can be understood from model studies is necessarily limited. Peptide-membrane interactions under physiological conditions are presumably influenced by additional factors such as: interactions with lipopolysaccharides, the presence of membrane proteins, membrane compositional heterogeneity, lipid domains, etc. In order to bridge the gap between the NMR studies of AMPs using model membranes and the AMP-membrane interactions occurring in intact cells, we have designed a procedure to incorporate high levels of 2H-NMR labels, specifically into the cell membrane, by creating a novel strain of E. coli: LA8. Using this strain we are able to reproducibly quantify the effects of the AMP MSI-78 on lipid chain order in bacterial membranes. Treatment with MSI-78 led to an increase in the disorder of the bacterial membrane. This was observed by the decrease in the average order parameter and by the increase in intensity at the lower frequencies. The peptide:lipid ratios needed to observe MSI-78's effects on acyl chain order in the intact cells falls between the ratios required to observe effects in NMR studies of model lipid systems and the ratios required to observe inhibition of cell growth in biological assays.

New capillary coatings in open tubular CEC as models for biological membranes

Novel stationary phases in open tubular CEC were investigated. The coating procedure was fast and simple. The coating material contained membrane suspension of different neuronal cell lines. The performance and stability of three cell lines: human neuroblastoma SH-SY5Y, murine microglia Bv-2 and human glioma U87-MG cells were studied. The coating solution was expected to contain both membrane proteins and membrane lipids. The presence of membrane proteins was tested by Western blotting and the presence of phospholipids by the analysis of phosphorus content. The stability of the coating was estimated by monitoring the mobility of EOF over successive runs. The effects of pH, storage time and temperature on the coating stability were also studied. The results showed that the cell membrane-based coating was stable over pH range of 6.5-8.5. Coatings derived from different cells yielded similar stability and EOF mobility. Capillary coated with a membrane solution was stable over 3-day period. The same coating solution could be used for 3 weeks.

Recent developments in fluorescence correlation spectroscopy for diffusion measurements in planar lipid membranes.

Fluorescence correlation spectroscopy (FCS) is a single molecule technique used mainly for determination of mobility and local concentration of molecules. This review describes the specific problems of FCS in planar systems and reviews the state of the art experimental approaches such as 2-focus, Z-scan or scanning FCS, which overcome most of the artefacts and limitations of standard FCS. We focus on diffusion measurements of lipids and proteins in planar lipid membranes and review the contributions of FCS to elucidating membrane dynamics and the factors influencing it, such as membrane composition, ionic strength, presence of membrane proteins or frictional coupling with solid support.

Continuum theory of a moving membrane

We derive a set of equations for the dynamics of evolving fluid membranes, such as cell membranes, in the presence of bulk fluids. We model the membrane as a surface endowed with a director field, which describes the local average orientation of the molecules on the membrane. A model for the elastic energy of a surface endowed with a director field is derived using liquid crystal theory. This elastic energy reduces to the well-known Helfrich energy in the limit when the directors are constrained to be normal to the surface. We then derive the full dynamic equations for the membrane that incorporate both the elastic and viscous effects, with and without the presence of bulk fluids. We also consider the effect of local spontaneous curvature, arising from the presence of membrane proteins. Overall, the systems of equations allow us to carry out stable, accurate, and robust numerical modeling for the dynamics of the membranes.

Neutrophil secretory defect in the gray platelet syndrome: A new case

We report the case of a 60-year-old woman who was newly diagnosed for the gray platelet syndrome (GPS). This patient had long-term thrombocytopenia which had been initially misdiagnosed as idiopathic thrombocytopenic purpura (ITP). Blood smear displayed characteristic gray platelets, allowing the diagnosis to be made, which was confirmed by electron microscopy (EM). Polymorphonuclear neutrophils (PMN) appeared poorly granulated on the May–Grunwald–Giemsa-stained blood smear. Flow cytometry analysis of PMN demonstrated increased expression of CD35, CD11b and CD18 at resting PMN surface, without any changes after fMLP stimulation. Ultrastructural study retrieved a decreased number of myeloperoxidase (MPO)-negative secondary granules in PMN. Immunolabeling confirmed the presence of membrane proteins and the absence of soluble content in platelet and megakaryocyte (MK) α-granules, and the decrease of secondary granules and secretory vesicles in PMN. This new observation demonstrates that the impairment of the secretory compartment of PMN is definitely a hallmark of GPS, and that the detection of these subtle abnormalities should be searched with adequate and up-to-date technical approaches.

A microfabricated device for subcellular organelle sorting.

We report a microfabricated field flow fractionation device for continuous separation of subcellular organelles by isoelectric focusing. The microdevice provides fast separation in very small samples while avoiding large voltages and heating effects typically associated with conventional electrophoresis-based devices. The basis of the separation is the presence of membrane proteins that give rise to the effective isoelectric points of the organelles. Simulations of isoelectric focusing of mitochondria in microchannels are used to assess design parameters, such as dimensions and time scales. In addition, a model of Joule heating effects in the microdevice during operation indicates that there is no significant heating, even without active cooling. The device is fabricated using a combination of photolithography, thin-film metal deposition/patterning, and electroplating techniques. We demonstrate that in the microfluidic devices, mitochondria from cultured cells migrate under the influence of an electric field into a focused band in less than 6 min, consistent with model predictions. We also illustrate separation of mitochondria from whole cells and nuclei as well as the separation of two mitochondrial subpopulations. When automated and operated in parallel, these microdevices should facilitate high-throughput analysis in studies requiring separation of organelles.

Anthrapyridones, a novel group of antitumour non-cross resistant anthraquinone analogues. Synthesis and molecular basis of the cytotoxic activity towards K562/DOX cells.

1. Multidrug resistance (MDR) to antitumour agents, structurally dissimilar and having different intracellular targets, is the major problem in cancer therapy. MDR phenomenon is associated with the presence of membrane proteins which belong to the ATP-binding cassette family transporters responsible for the active drug efflux leading to the decreased intracellular accumulation. 2. The search of new compounds able to overcome MDR is of prime importance. 3. Recently we have synthesized a new family of anthrapyridone compounds. The series contained derivatives modified with appropriate hydrophobic or hydrophylic substituents at the side chain. 4. The interaction of these derivatives with erythroleukemia K562 sensitive and K562/DOX resistant (overexpressing P-glycoprotein) cell lines has been examined. The study was performed using a spectrofluorometric method which allows to continuously follow the uptake and efflux of fluorescent molecules by living cells. 5. It was demonstrated that the increase in the lipophilicity of anthrapyridones favoured the very fast cellular uptake exceeding the rate of P-gp dependent efflux out of the cell. For these derivatives, very high accumulation (the same for sensitive and resistant cells) was observed and the in vitro biological data confirmed that these compounds exhibited comparable cytotoxic activity towards sensitive and P-gp resistant cell line. In contrast, anthrapyridones modified with hydrophylic substituents exhibited relatively low kinetics of cellular uptake. 6. For these derivatives decreased accumulation in resistant cells was observed and the in vitro biological data demonstrated that they were much less active against P-gp resistant cells in comparison to sensitive cells. 7. We also studied, using confocal microscopy, the intracellular distribution of anthrapyridones in NIH-3T3 cells. Our data showed that these compounds were strongly accumulated in the nucleus and lysosomes.

The Effects of Gramicidin on Electroporation of Lipid Bilayers

The effects of the channel-forming peptide gramicidin D (gD) on the conductance and electroporation thresholds of planar bilayer lipid membranes, made of the synthetic lipid 1-palmitoyl 2-oleoyl phosphatidylcholine (POPC), was studied. High-amplitude (∼200–900 mV) rectangular voltage pulses of 15 ms duration were used to perturb the bilayers and monitor the transmembrane conductance. Electroporation voltage thresholds were found, and conductance was recorded before and after electroporation. Gramicidin was added to the system in peptide/lipid ratios of 1:10,000, 1:500, and 1:15. The addition of gD in a ratio of 1:10,000 had no effect on electroporation, but ratios of 1:500 and 1:15 significantly increased the thresholds by 16% ( p < 0.0001) and 40% ( p < 0.0001), respectively. Membrane conductance before electroporation was measurable only after the addition of gD and increased monotonically as the peptide/lipid ratio increased. The effect of gD on the membrane area expansivity modulus ( K) was tested using giant unilamellar vesicles (GUVs). When gD was incorporated into the vesicles in a 1:15 ratio, K increased by 110%, consistent with the increase in thresholds predicted by an electromechanical model. These findings suggest that the presence of membrane proteins may affect the electroporation of lipid bilayers by changing their mechanical properties.

Immunolocalization of the sarcolemmal Ca2+/Mg2+ ecto-ATPase (myoglein) in rat myocardium.

Cardiac plasma membrane Ca2+/Mg2+ ecto-ATPase (myoglein) requires millimolar concentrations of either Ca2+ or Mg2+ for maximal activity. In this paper, we report its localization by employing an antiserum raised against the purified rat cardiac Ca2+/Mg2+ ATPase. As assessed by Western blot analysis, the antiserum and the purified immunoglobulin were specific for Ca2+/Mg2+ ecto-ATPase; no cross reaction was observed towards other membrane bound enzymes such as cardiac sarcoplasmic reticulum Ca(2+)-pump ATPase or sarcolemmal Ca(2+)-pump ATPase. On the other hand, the cardiac Ca2+/Mg2+ ecto-ATPase was not recognized by antibodies specific for either cardiac sarcoplasmic reticulum Ca(2+)-pump ATPase or plasma membrane Ca(2+)-pump ATPase. Furthermore, the immune serum inhibited the Ca2+/Mg2+ ecto-ATPase activity of the purified enzyme preparation. Immunofluorescence of cardiac tissue sections and neonatal cultured cardiomyocytes with the Ca2+/Mg2+ ecto-ATPase antibodies indicated the localization of Ca2+/Mg2+ ecto-ATPase in association with the plasma membrane of myocytes, in areas of cell-matrix or cell-cell contact. Staining for the Ca2+/Mg2+ ecto-ATPase was not cardiac specific since the antibodies detected the presence of membrane proteins in sections from skeletal muscle, brain, liver and kidney. The results indicate that Ca2+/Mg2+ ecto-ATPase is localized to the plasma membranes of cardiomyocytes as well as other tissues such as brain, liver, kidney and skeletal muscle.

Microtubule-associated Protein-dependent Binding of Phagosomes to Microtubules

In macrophages, phagosome movement is microtubule-dependent. Microtubules are a prerequisite for phagosome maturation because they facilitate interactions between phagosomes and organelles of the endocytic pathway. We have established an in vitro assay that measures the binding of purified phagosomes to microtubules. This binding depends on the presence of membrane proteins, most likely integral to the surface of phagosomes, and on macrophage cytosol. The cytosolic binding factor can interact with microtubules prior to the addition of phagosomes to the assay, suggesting that it is a microtubule-associated protein (MAP). Consistent with this, depletion of MAPs from the cytosol by microtubule affinity removes all binding activity. Microtubule motor proteins show no binding activity, whereas a crude MAP preparation is sufficient to support binding and to restore full binding activity to MAP-depleted cytosol. We show that the activating MAP factor is a heat-sensitive protein(s) that migrates at around 150 kDa by gel filtration.