New technologies have recently been developed for characterizing in vitro
MT dynamics at molecular scale resolution. Recent work by Kerssemakers et al
] first reported the dynamic growth and shortening behavior of MT plus-ends at the resolution of a single tubulin dimer (8 nm). Here, the authors held a bead/axoneme construct in an optical trap, and then allowed growth of a single MT into a rigid microfabricated barrier () [13
]. As the MT minus-end moved away from the barrier due to growth of the leading protofilament against the barrier, the bead position was displaced. By measuring the displacement of the bead in the trap over time, the authors were able to measure MT plus-end assembly dynamics of the leading protofilament at a sampling rate of 25 Hz and at a spatial resolution of 5-10 nm. Since previous studies using light microscopy reported MT plus-end dynamics at a spatial resolution of ~200 nm [5
], the results reported by Kerssemakers and co-workers provided an unprecedented look into MT plus-end dynamics at near molecular-level resolution.
Figure 2 Depiction of experimental assays for measuring various aspects of nanoscale MT dynamics. (A) The experimental assay designed by Kerssemakers et al  for measuring MT assembly dynamics at the nanoscale. A microtubule-attached bead (orange, bead not (more ...)
Interestingly, the relatively constant MT plus-end growth and shortening rates observed with light microscopy that have traditionally characterized “dynamic instability” behavior turn out to be highly variable at molecular scale resolution. Periods of rapid growth were observed that were clearly interspersed with periods of slower growth and even occasional shortening. Kerssemakers et al
] interpreted this rapid growth behavior as being “step-like”, in which rapid length changes were regularly interrupted with periods of slower changes in length. The addition of XMAP215, a microtubule-associated protein (MAP) known to increase the MT growth rate [15
], increased the frequency and duration of rapid MT plus-end length changes at the nanoscale. In addition, the step-like events appeared to be larger, on the order of tens of nm, which approaches the experimentally measured length of XMAP215 [17
]. Since the size of the steps in the absence of XMAP215 (20-30 nm) exceeded the size of a single subunit length (8 nm), it was suggested that small oligomers, composed of two or more subunits, can add to growing MT plus ends, and that addition of these oligomers is further promoted by XMAP215 (step size 40-60 nm). This study is highly significant because it demonstrated the feasibility of measuring MT assembly dynamics at near molecular-level resolution. This represented a resolution improvement of more than an order of magnitude in going from the light microscope (~200 nm), which has been the gold standard for observing MT assembly for the last 20 years, to the laser-tweezers-tracking method (5-10 nm).
More recently, Schek et al
] reported nanoscale MT dynamics using a similar experimental geometry to that of Kerssemakers et al
(). By collecting unfiltered data at higher temporal resolution (5 kHz), the authors were able to temporally average out thermal noise to report the molecular scale behavior of MT plus-end assembly at ~3.5 nm spatial resolution. In addition, the force due to polymerization at the MT plus-end was clamped to specific values by regular (10 Hz) adjustments to the laser trap position. Thus, leading protofilament MT plus-end growth could be characterized at higher temporal and spatial resolution and under conditions of relatively constant, low compressive load.
Similar to results reported by Kerssemakers et al
], Schek et al
] reported highly variable MT plus-end growth behavior. Here, rapid advances in plus-end polymerization were balanced by periods of slow growth and, surprisingly, shortening excursions of ~40 nm or more that did not result in plus-end catastrophe. In this work, the improved temporal resolution of the experiment showed that periods of rapid growth, that could appear step-like at lower resolution, were in fact made up of a series of small (nm) increments. In over 16,000 observations, there were not any events that corresponded to the 20-30 nm steps suggested by Kerssemakers et al
], demonstrating that oligomer-mediated addition was extremely rare under the conditions of the Schek et al
While Schek et al
's experiments allowed direct measurement of characteristics such as nanoscale changes in leading protofilament length, they also provided more indirect but equally important insight into the size and structure of the stabilizing GTP cap. A long-standing view in the literature is that the GTP-cap could be as thin as a single layer [19
]. However, frequent nanoscale shortening events of 40 nm or more (~5 tubulin subunit layers) without MT catastrophe suggested a more substantial stabilizing GTP-cap depth. To interpret these fluctuations, Schek et al
compared the observed dynamics to those predicted by a GTP-cap model that assumes first-order hydrolysis once a GTP-tubulin is buried in the lattice [24
]. Here, an approximately exponentially distributed GTP-cap, typically composed of ~40-60 GTP-tubulins, was sufficient to explain the substantial nanoscale shortening events observed during MT plus-end growth.
Finally, by allowing for regular adjustments to the bead laser trap position such that nearly constant compressive loads were maintained at the growing MT tip, Schek et al
reported the effect of increased compressive load on nanoscale MT growth behavior. Interestingly, the variability in nanoscale growth behavior was suppressed at higher compressive forces as compared to low force behavior, an effect that was predicted by computational modeling [24
]. Using the model as a guide, it was hypothesized that leading protofilaments have greater difficulty remaining ahead of lagging protofilaments when the compressive force is relatively large and the probability of subunit addition to leading protofilaments is therefore relatively small. Because lagging protofilaments stay closer to the leading protofilament, the shortening that occurs when the leading protofilament depolymerizes is decreased at higher compressive forces. As a result, the amplitude of both nanoscale growth and shortening excursions during assembly under load is suppressed at higher force. The overall consequence is that the net growth rate is only weakly reduced by load (up to 2.5 pN). In summary, the study by Schek et al
] showed that a single-layer GTP-cap model is very unlikely, that MT growth by oligomers is very unlikely, and that net growth is only weakly slowed by compressive load. Together, the studies of Kerssemakers et al
and Schek et al
open new doors for MT research, where MAPs and drugs can be added and the effects observed at near molecular resolution. An important aspect of these efforts will be the integration of computational models for MT assembly in the presence of MAPs and drugs, whose predictions can be directly compared to these experiments.