Microtubules (MTs) are highly dynamic components of the cell cytoskeleton that are necessary for many functions, including cell division, cellular locomotion, and intracellular transport. An essential mechanistic feature of MT physiology is dynamic instability, which is characterized by the frequent polymerization and depolymerization of tubulin subunits at MT ends. This dynamicity is critical to MT function and is regulated by MT-associated proteins (MAPs), which interact with tubulin dimers and/or the MTs themselves. Tau is a prominent neuronal MAP that stabilizes MTs by promoting growth events, stability, and by suppressing shortening events. On the other hand, dysregulation and mutation of tau are associated with pathogenesis in various neurodegenerative diseases, such as Alzheimer’s disease (AD), frontotemporal dementia with Parkinsonism-17 (FTDP-17), and progressive supranuclear palsy (PSP). Taken together, it is critical to understand both normal tau physiology as well as how altered tau function leads to disease pathogenesis. Previous research has suggested that tau is able to dimerize or oligomerize via its N-terminal projection domain as part of its normal function. One currently proposed model, based on in vitro data, is that two tau molecules form an “electrostatic zipper” in which the N-termini of the two molecules associate in an antiparallel fashion, with the C-termini containing the MT-binding region of each tau molecule extending away from one another. If correct, this model could explain many features of tau action. We investigated this hypothesis in mammalian cells using a split-luciferase strategy in order to (i) test the above stated model for tau oligomerization in cells and (ii) identify and map regions of the protein that are capable of tau-tau oligomerization. We found that constructs containing the N-terminus of tau produce significantly higher luciferase signals indicative of oligomerization compared to constructs containing the C-terminus. More specifically, the construct containing amino acids 1-120 produces the strongest luciferase signal, consistent with our proposed model that the N-terminus of tau is responsible, at least in part, for its oligomerization activity. Interestingly, C-terminal regions of tau are also capable of promoting tau oligomerization. Taken together, our data suggest that both the N- and C- termini of tau are each sufficient to promote tau oligomerization in mammalian cells.

Microtubules (MTs) are dynamic cytoskeletal polymers that are essential for many cellular processes, including cell division, maintenance of cellular shape, intracellular transport, and cell signaling. Their dynamic, switch-like behavior, known as MT dynamic instability, arises from the conformational changes associated with GTP hydrolysis by β-tubulin, but exactly how the tubulin conformational cycle contributes to the overall MT dynamic state remains unclear. Proper regulation of MT dynamicity is critical across almost all cell types, but their necessity for proper chromosome alignment and division during mitosis make them an especially effective target for anti-cancer drugs targeting rapidly dividing tumor cells. Despite the broad use of these MT-targeting agents (MTAs) as chemotherapeutics, we often lack understanding of the mechanisms of action underlying the global changes they enact on MT dynamics. We investigated the binding of MTA Monomethyl auristatin E (MMAE), as a free drug or as an antibody-drug conjugate (ADC), to MTs and free tubulin subunits, and characterized its effects upon MT dynamics and MT morphology as well as cell proliferation, cell cycle regulation, and the generation of mitotic spindle abnormalities in cultured human cells. In combination with comparisons made to other MTAs, our data provide further insights into the molecular mechanisms underlying normal MMAE action as well as those governing MMAE ADC-induced peripheral neuropathy.

In cells, MT-associated proteins (MAPs) help to regulate MT dynamics. Tau is a neuronal MAP that regulates the critical growing and shortening behaviors of neuronal MTs, and its normal activity is essential for neuronal development and maintenance. Accordingly, aberrant tau action is tightly associated with Alzheimer's disease and is genetically linked to several additional neurodegenerative diseases known as tauopathies. Indeed, one often suggested model for pathological tau action in Alzheimer's and related, dementia-causing tauopathies is the destabilization of axonal MTs, leading to aberrant axonal transport and neuronal cell death. Although tau's most well-characterized activity is its promotion of net MT growth and stability, the precise mechanistic details governing its regulation of MT dynamics remain unclear.

We used the slowly-hydrolyzable GTP analog, guanylyl-(α,β)-methylene-diphosphonate (GMPCPP), to examine the structural effects of tau at MT ends that may otherwise be too transient to observe. We found that co-incubation of GMPCPP tubulin and tau resulted in the formation of extended, multiprotofilament-wide tubulin spirals emanating i) from the ends of pre-assembled MTs at 25 ◦C, ii) from free tubulin heterodimers at 4 ◦C, and iii) from free tubulin heterodimers at 34 ◦C. While 3R and 4R tau isoforms promoted MT assembly intermediates similarly, 4R tau stabilized disassembly spiral intermediates much more effectively than 3R tau, consistent with 4R tau's more effective suppression of MT shortening events. Importantly, all of these spiral structures were also observed, albeit at much lower frequencies, in the absence of tau, and have also been observed in previous studies of both GTP and GMPCPP tubulin, consistent with the notion that that they are bona fide intermediates in the MT assembly/disassembly processes.

Finally, three tau proteins harboring mutations that cause neurodegeneration and dementia were differentially compromised in their abilities to stabilize intermediate structures. Taken together, we propose that tau promotes the formation/stabilization of intermediate states in MT assembly and disassembly by promoting both longitudinal and lateral tubulin-tubulin contacts. We hypothesize that these activities represent fundamental aspects of tau action that normally occur at the GTP-rich ends of GTP/GDP MTs and that may be compromised in neurodegeneration-causing tau variants.