Mysterious Tau Hot Spots Speckle Plasma Membrane

Once thought of as simply a microtubule-binding protein, tau is now known to transit through several cellular locations, including the plasma membrane, where it can slip out into extracellular space. This getaway may enable tau aggregates to pass between neurons. Still, how tau interacts with the membrane remains poorly understood. In the August 25 EMBO Journal, researchers led by Jürgen Götz at the University of Queensland, Brisbane, Australia, reported that tau congregates at the plasma membrane, at least in cultured cells.

  • In cultured cells, tau forms transient hot spots at the cell membrane.
  • These clusters mostly stay in place and last about 12 seconds.
  • Do they facilitate tau aggregation, secretion?

Real-time super-resolution microscopy revealed formation of tau clusters that lasted for several seconds, and sometimes moved along the membrane before dispersing again. The scientists do not know how these mysterious hot spots form, or what their purpose might be, if any. Potentially, they could serve as stepping stones to the aggregation and secretion of tau, they suggested.

Stuck? At the plasma membrane, tau sometimes moves freely (gray lines), but other times seems confined (gold), or does not move at all (red), suggesting the molecules are bound to the lipid bilayer. [Courtesy of Padmanabhan et al., The EMBO Journal.]

"The paper adds a new dimension to our understanding of tau’s distribution in the cell, and it demonstrates the power of super-resolution techniques in revealing new biology,” William McEwan at the University of Cambridge, U.K., wrote to Alzforum (full comment below).

In animal models, aggregates of tau appear to travel along neuronal connections, passing between cells and spreading through the brain (Jun 2009 news; Dec 2018 conference news). In cultures, this cellular hand-off has been directly observed (Mar 2013 conference news; May 2018 news). Synaptic activity may affect this process, because neurons secrete tau in response to stimulation (Feb 2014 news; Jun 2016 news). But does tau gather at the membrane before escaping a cell?

To find out, first author Pranesh Padmanabhan tagged human tau with a fluorescent protein, expressed it in a mouse neuroblastoma cell line, then followed it using a type of super-resolution microscopy called single-particle tracking photoactivated localization microscopy.

Narrow Focus. To detect membrane-associated tau, researchers tagged the 4R isoform with a photoactivatable label, then used the reflective properties of the glass coverslip to confine light to this surface and excite only those fluorophores in contact with it, i.e., at the plasma membrane. [Courtesy of Padmanabhan et al., The EMBO Journal.]

Padmanabhan detected three different types of tau near the plasma membrane: immobile, confined, and freely moving tau. Confined tau moved within a radius of about 90 nm, immobile tau less than 70 nm. Further analysis showed that these restricted tau molecules were clumping into spots about 140 nm wide, which lasted from two to 20 seconds (see image below). Some of these hot spots were completely stationary, while others slid slightly. The results suggest that tau might be clustering with proteins or lipids in the plasma membrane, the authors noted.

Home in on Hot Spot From the raw fluorescence image of a cell (left), scientists zoomed in (dotted square) to generate an intensity map (middle left) of fluorescence, then mapped that using a Voronoi diagram (middle right) to reveal tau hot spots (right). [Courtesy of Padmanabhan et al., The EMBO Journal.]

Are these hot spots regulated? Padmanabhan tested tau variants that poorly bind microtubules, freeing them up for other functions. He found no change in the pattern of the hot spots. Likewise, directly disrupting microtubules boosted the amount of free tau in the cell, but did not affect the clusters.

Since cholesterol has been found to hamper tau secretion and facilitate tau uptake in cell culture, the authors lowered levels of the lipid by adding the sugar methyl-β-cyclodextrin, which solubilizes cholesterol and removes it from the cell membrane (Merezhko et al., 2018; May 2022 news). Again, the hot spots were indifferent. Immortalized cells do not always reflect what happens in brain. However, tau formed the same type of clusters in HEK293T kidney cells, and in neuroblastoma cells labeled with a different tag, showing that this behavior was not dependent on cell type or fluorescent domain.

Götz told Alzforum that tau hot spots likely represent a physiological phenomenon, since they occurred in healthy cells expressing wild-type tau. Nonetheless, he believes that under pathological conditions, these clusters might facilitate tau aggregation and release. In future work, he will examine what controls these structures, what they do, and whether their behavior changes in the presence of disease-associated proteins, such as Aβ oligomers or mutant tau.

Tsuneya Ikezu at the Mayo Clinic in Jacksonville, Florida, suggested investigating which domain of tau binds the plasma membrane, and whether the same structures form in neurons under more physiological conditions using endogenous tau expression. McEwan agreed, “An understanding of the molecular requirements for formation of the hotspots could be very revealing, and a good place to start for future studies.”—Madolyn Bowman Rogers

Comments

  1. These are very interesting observations revealing the presence of transient tau accumulations that form at, or very near, the plasma membrane. It remains to be determined whether these structures result from tau’s ability to interact with lipids, or arise via protein-protein interaction (or some combination of both).

    As the authors note, the results instantly raise a lot of questions as to the functional relevance of these structures. What is the relationship between the hotspots and fibrillar tau aggregation? Could these local densities promote the conversion of tau from native soluble protein to filamentous aggregates? And is there a link between these tau assemblies and the unconventional secretion of tau?

    An understanding of the molecular requirements for formation of the hotspots could be very revealing, and a good place to start for future studies. Investigating the requirements in tau might provide important information to the role of the structures. For instance, if known modifiers of tau secretion, such as mutation of its cysteine residues (Katsinelos et al., 2021), were to impact hotspot formation, it would suggest that the two phenomena are linked. Determining whether the hotspots are observed with protein-free lipid vesicles would help determine the nature of the interaction.

    Finally, determining whether the hotspot structures are present in neural tissue is important to understand their relevance to the physiological condition. The paper adds a new dimension to our understanding of tau’s distribution in the cell and demonstrates the power of super-resolution techniques in revealing new biology.

    References:

    Katsinelos T, McEwan WA, Jahn TR, Nickel W. Identification of cis-acting determinants mediating the unconventional secretion of tau. Sci Rep. 2021 Jun 21;11(1):12946. PubMed.

    View all comments by William McEwan

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References

News Citations

  1. Traveling Tau—A New Paradigm for Tau- and Other Proteinopathies?
  2. Toxic Tau: Who Are You, and Where Are You From?
  3. Tau, α-Synuclein Spread: Crazy Stuff—How Might It Work?
  4. To Deliver Itself From Cell to Cell, Phospho-Tau Uses UPS
  5. Neurons Release Tau in Response to Excitation
  6. Excited Neurons Release More Aberrant Tau
  7. Membrane Border Patrol: Cholesterol Stymies Tau Uptake, Aggregation

Paper Citations

  1. Merezhko M, Brunello CA, Yan X, Vihinen H, Jokitalo E, Uronen RL, Huttunen HJ. Secretion of Tau via an Unconventional Non-vesicular Mechanism. Cell Rep. 2018 Nov 20;25(8):2027-2035.e4. PubMed.

Further Reading

Primary Papers

  1. Padmanabhan P, Kneynsberg A, Cruz E, Amor R, Sibarita JB, Götz J. Single-molecule imaging reveals Tau trapping at nanometer-sized dynamic hot spots near the plasma membrane that persists after microtubule perturbation and cholesterol depletion. EMBO J. 2022 Oct 4;41(19):e111265. Epub 2022 Aug 25 PubMed.

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