Mechanistic insights into the regulation of microtubule assembly and dynamic instability by tau and MMAE

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