A bacteriophage tubulin harnesses dynamic instability to center DNA in infected cells
- University of California, San Diego, United States
- Howard Hughes Medical Institute, University of California, San Francisco, United States
Figures
Figure 1
TIRF microscopy reveals polarity and dynamic instability in PhuZ filaments.
(A) Cy3 labeled PhuZ filaments exhibit treadmilling in the presence of GTP. Many filaments displayed non-uniform intensity (highlighted by arrows), which we used as stationary points to monitor …
Figure 2
Nucleotide hydrolysis destabilizes PhuZ filaments.
(A) Right-angle light scattering traces of D190A-PhuZ polymerization at 1.5 (purple), 2 (green), 2.5 (red), and 3 (blue) μM upon addition of 1 mM GTP. (B) Right-angle light scattering traces of PhuZ …
Figure 3
PhuZ filaments are distributed throughout the cell in the absence of phage.
(A) GFP-PhuZ expressed by itself (in the absence of phage infection) assembles relatively short filaments in P. chlororaphis. Four individual cells (outlined) with multiple filaments are shown. (B) …
Figure 4
PhuZ forms a spindle composed of bipolar filaments in vivo.
(A) P. chlororaphis cells (outlined) that have been infected with phage 201ϕ2-1 for approximately 60 min have bipolar GFP-PhuZ spindles. (B) Kymograph of GFP-PhuZ fluorescence intensity in a single …
Figure 5
Filaments of the PhuZ spindle are dynamically unstable in vivo.
(A) Time-lapse sequence showing a single cell with a bipolar spindle over the course of 129 s. The filaments of the spindle can be observed to grow and shrink (arrows and Video 5). (B–D) Length …
Figure 6
Fluorescence in situ hybridization (FISH) during phage infection.
(A) Infected P. chlororaphis cells were fixed and hybridized with DNA probes specific for either the host chromosomal DNA (pink, labeled with Cy3) or phage 201ϕ2-1 DNA (green, labeled with Cy3). …
Figure 7
Observation of phage nucleoid migration by time-lapse microscopy.
(A) Time-lapse sequence showing development of a phage nucleoid (arrows) in a single cell over the course of 63 min. Membranes are stained red with FM 4-64 and the DNA is stained green with Syto16. …
Figure 8
Infection Nucleoid Centering is Independent of DNA replication.
(A) DNA content of infection nucleoids was measured by DAPI staining. Cells were grown on an agarose pad and infected with phage 201ϕ2-1. Ciprofloxacin was applied to cells 10 min prior to infection …
Figure 9
Model of PhuZ bipolar spindle formation during lytic growth.
PhuZ is expressed early in lytic growth and forms dynamically unstable polymers anchored at the cell poles. Filaments polymerize unidirectionally, with GTP-bound (green) subunits adding to one end, …
Videos
Video 1
TIRF microscopy of 2.5 μM Cy3-PhuZ (20% Cy3) filaments reveals PhuZ filaments are dynamic and translocate around the field of view. Filaments treadmill and undergo catastrophic depolymerization. …
Video 2
Close up of Cy3-labelled PhuZ showing annealing, severing, and depolymerization events. Images were acquired every 500 ms for 100 s. Scale bar equals 2 μm.
Video 3
Close up of Cy-3 labelled PhuZ filaments demonstrating a full depolymerization event. Zoom in from Video 1. Scale bar equals 2 μm.
Video 4
Dynamic instability and polarity of PhuZ filaments revealed by two-color TIRF microscopy. GMPCPP stabilized PhuZ seeds (20% Cy5, 5% biotin, red) were attached to biotin-PEG coated glass, and 1.5 μM …
Video 5
Time-lapse movie of P. chlororaphis cells expressing GFP-PhuZ and infected with phage 201φ2-1 shows that PhuZ filaments form a spindle in which the centrally located ends of the filaments display …
Video 6
Time-lapse movie corresponding to the cell in Figure 4A showing development of a phage nucleoid in a single infected cell over the course of 63 min. The phage nucleoid first appears as a small green …
Video 7
Time-lapse movie showing development of a phage nucleoid in a single infected cell over the course of 49 min. This example of nucleoid formation and movement corresponds to one of the nucleoids …
Tables
Table 1
In vitro polymerization parameters
| Pol. Rate (µm/min*µM) | Plus-end Depol. Rate (µm/min) | Minus-end Depol. Rate (µm/min) | |
|---|---|---|---|
| PhuZ | 1.9 ± 0.1 (n = 40) | 108 ± 20 (n = 40) | 15 ± 5 (n = 40) |
| Mammalian tubulin | 0.2 (Hyman et al., 1992) | 13 (Walker et al., 1988) | 7.5 (Mitchison and Kirschner, 1984a) |
| Yeast tubulin | 0.18 (Gupta et al., 2002; Bode et al., 2003) | 103 (Gupta et al., 2002; Bode et al., 2003) | Not measured |
