Microtubule Dynamics Reconstituted In Vitro and Imaged by Single-Molecule Fluorescence Microscopy

Microtubules, the dynamic, yet stiff hollow tubes built from αβ-tubulin protein heterodimers, are thought to be present only in eukaryotic cells. Here, we report a 3.6-Å helical reconstruction electron cryomicroscopy structure of four-stranded mini microtubules formed by bacterial tubulin-like Prosthecobacter dejongeii BtubAB proteins. Despite their much smaller diameter, mini microtubules share many key structural features with eukaryotic microtubules, such as an M-loop, alternating subunits, and a seam that breaks overall helical symmetry. Using in vitro total internal reflection fluorescence microscopy, we show that bacterial mini microtubules treadmill and display dynamic instability, another hallmark of eukaryotic microtubules. The third protein in the btub gene cluster, BtubC, previously known as “bacterial kinesin light chain,” binds along protofilaments every 8 nm, inhibits BtubAB mini microtubule catastrophe, and increases rescue. Our work reveals that some bacteria contain regulated and dynamic cytomotive microtubule systems that were once thought to be only useful in much larger and sophisticated eukaryotic cells.

Section: Methodsmentioning

confidence: 99%

Four-stranded mini microtubules formed by Prosthecobacter BtubAB show dynamic instability

Deng

Fink

Bharat

et al. 2017

“…Gliding motility assays were performed in flow chambers as described previously (57). In short, a flow chamber was assembled from a PEG-functionalized 22-× 22-mm coverslip and a 10-× 10-mm Si chip featuring an oxide layer of about 30 nm coated with dichlorodimethylsilane (Sigma-Aldrich) using a coating procedure described previously (58). Spacers of NESCO film were placed such that the resulting channels were 10 mm × 1.5 mm × 100 μm.…”

Section: Methodsmentioning

confidence: 99%

Working stroke of the kinesin-14, ncd, comprises two substeps of different direction

Nitzsche

Dudek

Hajdo

et al. 2016

Self Cite

Single-molecule experiments have been used with great success to explore the mechanochemical cycles of processive motor proteins such as kinesin-1, but it has proven difficult to apply these approaches to nonprocessive motors. Therefore, the mechanochemical cycle of kinesin-14 (ncd) is still under debate. Here, we use the readout from the collective activity of multiple motors to derive information about the mechanochemical cycle of individual ncd motors. In gliding motility assays we performed 3D imaging based on fluorescence interference contrast microscopy combined with nanometer tracking to simultaneously study the translation and rotation of microtubules. Microtubules gliding on ncd-coated surfaces rotated around their longitudinal axes in an [ATP]-and [ADP]-dependent manner. Combined with a simple mechanical model, these observations suggest that the working stroke of ncd consists of an initial small movement of its stalk in a lateral direction when ADP is released and a second, main component of the working stroke, in a longitudinal direction upon ATP binding.kinesin | microtubule | gliding motility assay | fluorescence interference contrast microscopy | mathematical modeling S ingle-molecule techniques, in particular single-molecule fluorescence (1) and optical tweezers (2), have greatly advanced our understanding of motor proteins. Such experiments normally rely on the processivity of the motor, which makes it possible to observe multiple consecutive working cycles (i.e., steps). Although single-molecule mechanical measurements on nonprocessive motors can be performed with optical tweezers (3, 4), they require a sophisticated statistical analysis (5, 6) and, if the motor is to be studied under load, advanced feedback (7) or force-clamp mechanisms (8). The minus-end directed C-terminal kinesin-14, ncd, is such a nonprocessive motor. It has been shown to play an essential role in spindle focusing (9-11) and to slide and cross-link microtubules (12, 13).For the working stroke of a single ncd motor a lever arm model has been suggested (14, 15). However, as reviewed in ref. 16 the triggering event for the working stroke is still under debate. On the one hand, it has been suggested that the working stroke is connected to the ADP release (14, 17). On the other hand, it has been proposed to be coupled to the binding of ATP to the microtubule (MT)-ncd complex (4,15,18). In another study, a twostage displacement of the ncd stalk has been proposed where stalk displacement is initiated by binding of ncd to the MT and completed upon ATP binding (19). However, even though a variety of approaches including X-ray crystallography (15), EM (20-23), optical trapping (4, 24), FRET (19), and computational modeling (25) have been used to study the coupling between nucleotide states and mechanical states of ncd, the exact mechanism remains to be elucidated.Alternatively, nonprocessive motors can be studied in gliding motility assays (26) albeit with the disadvantage that single-motor behavior is difficult to access. On an ncd...

“…Microscope chambers were constructed of silanized coverslips as described previously (34,35). Channels were incubated with antibiotin antibody (Sigma) in phosphatebuffered saline (PBS) for 3 min, washed with 40 μL of PBS, and blocked for 30 min with 1% Pluronic F127 (Sigma) in PBS.…”

Section: Methodsmentioning

confidence: 99%

Reconstitution of a prokaryotic minus end-tracking system using TubRC centromeric complexes and tubulin-like protein TubZ filaments

Fink

Löwe

2015

Segregation of DNA is a fundamental process during cell division. The mechanism of prokaryotic DNA segregation is largely unknown, but several low-copy-number plasmids encode cytomotive filament systems of the actin type and tubulin type important for plasmid inheritance. Of these cytomotive filaments, only actin-like systems are mechanistically well characterized. In contrast, the mechanism by which filaments of tubulin-like TubZ protein mediate DNA motility is unknown. To understand polymer-driven DNA transport, we reconstituted the filaments of TubZ protein (TubZ filaments) from Bacillus thuringiensis pBtoxis plasmid with their centromeric TubRC complexes containing adaptor protein TubR and tubC DNA. TubZ alone assembled into polar filaments, which annealed laterally and treadmilled. Using single-molecule imaging, we show that TubRC complexes were not pushed by filament polymerization; instead, they processively tracked shrinking, depolymerizing minus ends. Additionally, the TubRC complex nucleated TubZ filaments and allowed for treadmilling. Overall, our results indicate a pulling mechanism for DNA transport by the TubZRC system. The discovered minus end-tracking property of the TubRC complex expands the mechanistic diversity of the prokaryotic cytoskeleton.cytoskeleton | tubulin homologue | DNA segregation