Porcine brain tubulin (T240-DX) was run on a 4-20% SDS PAGE system and stained with 0.1% Coomassie Blue.  The gel in figure 3 shows 50 mg of tubulin protein.  Scanning densitometry determined the protein to be >99% pure tubulin.

 Brief Description Of The Three Phases Of Tubulin Polymerization:

 This section is designed to familiarize the reader with the  phase of polymerization.  The graph shown below represents a generalized tubulin polymerization curve.

Figure legend: The graph depicts the recognized phases of microtubule polymerization, as assayed by turbidity measurements at OD340.  These are the lag phase (phase I), the elongation phase (phase II) and the steady state phase (phase III). These phases can be seen clearly in a control reaction such as that for line number 2.

The Lag Phase:        The first thing necessary to achieve microtubule polymerization is the presence of nucleation site/s.  As the name suggests, this is simply a site from which microtubule polymerization can occur.  A nucleation site in the cell for example would be a centrosome or an axoneme.  In the in vitro reactions that we have used for these drug assays the tubulin must create its own nucleation sites by forming small oligomers from which larger microtubule polymers can grow.  Formally, in vitro nucleation can be defined as the formation of early oligomers, up to the point where their association rate constants become independent of their size.  Because the oligomeric nucleation sites are very small, and there are not large numbers of them initially, they will not scatter light to any appreciable degree and hence the resultant lack of any turbidity reading during this lag phase.

 Many factors can affect the duration of a polymerization lag phase.  Foremost among these is the critical concentration of the polymerization reaction (defined as the concentration of tubulin heterodimer necessary to allow polymerization to occur).  The critical concentration of tubulin in a polymerization reaction is greatly dependent upon the experimental conditions.  The table below illustrates this phenomenon;

Experimental Condition                                                   Estimated Critical Concentration of Tubulin     

It is clear from the above description that any compound affecting the critical concentration of tubulin under a given set of experimental conditions may affect the nucleation (lag phase) of the polymerization reaction.  It should also be remembered that, in certain cases, the critical concentration for nucleation is higher than the critical concentration for microtubule elongation.  Nucleation is determined in the following analysis by measuring the Onset time. This is the time taken to pass 1/10^th of the control’s maximum optical density. For example: with a maximum control OD340nm of 0.10, the Onset time will be equal to the time when the polymerization profile passes 0.01 OD340nm.

The Growth Phase:  Nucleation, assembly and disassembly are the three events that contribute towards this phase of polymerization.   If turbidity analysis indicates that a compound is affecting the growth phase, one can then manipulate polymerization conditions in order to examine the assembly and/or disassembly reactions separately.   These experiments would begin to address the mechanism of action of the compound.  For example, to examine growth (assembly/disassembly) rate independently of nucleation one can introduce seeds into the tubulin solution prior to polymerization.  One can selectively examine disassembly by creating conditions in which both nucleation and assembly are negligible.  This is most frequently accomplished by extensive dilution of the polymerization reaction to below the critical concentration. Growth rate is determined in the following analysis by measuring the polymerization rate in mOD340nm units per min. The highest rate (Vmax) is utilized for this calulation.

The Steady State Phase:                The steady state phase is established when the free heterodimer concentration is equal to the critical concentration for polymerization.  Hence the polymer mass concentration remains essentially constant even though heterodimers are continuously being taken up in the polymer and dissociated from the ends.  Any compound that lowers the critical concentration for polymerization will result in the production of a greater polymer mass and a concomitant increase in the turbidity measurement at steady state.  The steady state is obtained from the following analysis by measuring the maximum optical density. By comparing this to the control’s maximum optical density we can determine whether the samples show different activity.

Significance Analysis:                    Significance is determined by the additive affects of all three phases. If the samples are more than 20% different from the control, then they are said to be significant and assigned a value of one. If they are not different they are assigned a value of zero. All three phases are added together to come up with an overall significance. See the last column in Analyzed data to see the significance values assigned to each test.

The Microtubule Network As A Target For Therapeutic Agents:

Microtubules are formed by the polymerization of the heterodimeric protein tubulin.  There are at least 14 tubulin isotypes in higher eukaryotes, some of which are expressed in a tissue specific manner.  Observation of microtubule polymerization in vitro led to the discovery that microtubules are intrinsically dynamic structures that are continually in a state of flux.  This property, termed dynamic instability, was subsequently shown to occur in vivo.  Importantly, microtubule dynamics have been shown to alter during the cell cycle and are often very different between cell types.  The dynamic nature of microtubules is now recognized to be essential for them to mediate their various cellular functions.    Agents that can selectively disrupt  microtubule dynamics, either by targeting a specific tubulin isotype or a particular stage of cell division would be predicted to have great therapeutic value.  Several highly promising anti-cancer drugs (which exploit the difference in microtubule dynamics between rapidly dividing (cancerous) and predominantly non-dividing (most normal cell populations) cells have been developed that act by disrupting microtubule dynamics.  These include microtubule de-stabilizing drugs such as vinblastine and the stabilizing drug taxol.

Introduction To The In Vitro Assay Used In These Studies:

In order to assess a compound's potential to effect tubulin polymerization one must develop a suitable assay.  One such assay follows polymerization in vitro by measuring the increase in absorbance at 340 nm.  The effect of a drug upon polymerization is reflected as an alteration in the turbidity measurements.  

The intrinsic ability of pure tubulin to polymerize is very much a function of the experimental conditions.  Under a given set of conditions a polymerization curve will reflect the three major phases of the polymerization event, namely nucleation (lag phase), growth (growth phase) and steady state (steady state phase).  However, if one has reason to believe that the compound of interest is having a major effect during a specific phase then one can manipulate the polymerization conditions so as to favor a particular phase.  The assay conditions that we have selected generate a polymerization reaction that is relatively sensitive to both stabilizing and destabilizing factors.  This is the assay of choice when the effect of a compound cannot be predicted, as in the preliminary screen of a novel compound.  Clearly, the turbidity assay is very versatile and it has been used to identify and characterize many microtubule binding compounds.